Methods and compositions for optimization of oxygen transport by cell-free systems

ABSTRACT

Compositions, and methods of use thereof, for use as blood substitute products comprise aqueous mixtures of oxygen-carrying and non-oxygen carrying plasma expanders and methods for the use thereof. The oxygen-carrying component may consist of any hemoglobin-based oxygen carrier, while the non-oxygen carrying plasma expander my consist of any suitable diluent.

The present application is a Continuation-in-Part of U.S. patentapplication Ser. No. 08/810,694, filed Feb. 28, 1997.

This invention was made with Government support under the NationalInstitutes of Health (NIH) awarded by contract P01 HL48018. TheGovernment has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates generally to blood products, and moreparticularly to compositions comprising mixtures of oxygen-carrying andnon-oxygen carrying plasma expanders and methods for their use.

BACKGROUND OF THE INVENTION

A. The Circulatory System and the Nature of Hemoglobin

The blood is the means for delivering nutrients to the tissues andremoving waste products from the tissues for excretion. The blood iscomposed of plasma in which red blood cells (RBCs or erythrocytes),white blood cells (WBCs), and platelets are suspended. Red blood cellscomprise approximately 99% of the cells in blood, and their principalfunction is the transport of oxygen to the tissues and the removal ofcarbon dioxide therefrom. The left ventricle of the heart pumps theblood through the arteries and the smaller arterioles of the circulatorysystem. The blood then enters the capillaries, where the majority of theexchange of nutrients and cellular waste products occurs. (See, e.g., A.C. Guyton, Human Physiology And Mechanisms Of Disease (3rd. ed.; W.B.Saunders Co., Philadelphia, Pa.), pp. 228-229 [1982]). Thereafter, theblood travels through the venules and veins in its return to the rightatrium of the heart. Though the blood that returns to the heart isoxygen-poor compared to that which is pumped from the heart, in restingman the returning blood still contains about 75% of the original oxygencontent.

The reversible oxygenation function (i.e., the delivery of oxygen andthe removal of carbon dioxide) of RBCs is carried out by the proteinhemoglobin. In mammals, hemoglobin has a molecular weight ofapproximately 68,000 and is composed of about 6% heme and 94% globin. Inits native form, it contains two pairs of subunits (i.e., it is atetramer), each containing a heme group and a globin polypeptide chain.In aqueous solution, hemoglobin is present in equilibrium between thetetrameric (MW 68,000) and dimeric forms (MW 34,000); outside of theRBC, the dimers are prematurely excreted by the kidney (plasma half-lifeof approximately two to four hours). Along with hemoglobin, RBCs containstroma (the RBC membrane), which comprises proteins, cholesterol, andphospholipids.

B. Exogenous Blood Products

Due to the demand for blood products in hospitals and other settings,extensive research has been directed at the development of bloodsubstitutes and plasma expanders. A blood substitute is a blood productthat is capable of carrying and supplying oxygen to the tissues. Bloodsubstitutes have a number of uses, including replacing blood lost duringsurgical procedures and following acute hemorrhage, and forresuscitation procedures following traumatic injury. Plasma expandersare blood products that are administered into the vascular system butare typically not capable of carrying oxygen. Plasma expanders can beused, for example, for replacing plasma lost from burns, to treat volumedeficiency shock, and to effect hemodilution (for, e.g., the maintenanceof normovolemia and to lower blood viscosity). Essentially, bloodproducts can be used for these purposes or any purpose in which bankedblood is currently administered to patients. (See, e.g., U.S. Pat. No.4,001,401 to Bonson et al. and U.S. Pat. No. 4,061,736 to Morris et al.,hereby incorporated by reference).

The current human blood supply is associated with several limitationsthat can be alleviated through the use of an exogenous blood product. Toillustrate, the widespread availability of safe and effective bloodsubstitutes would reduce the need for banked (allogeneic) blood.Moreover, such blood substitutes would allow the immediate infusion of aresuscitation solution following traumatic injury without regard tocross-matching (as is required for blood), thereby saving valuable timein resupplying oxygen to ischemic tissue. Likewise, blood substitutescan be administered to patients prior to surgery, allowing removal ofautologous blood from the patients which could be returned later in theprocedure, if needed, or after surgery. Thus, the use of exogenous bloodproducts not only protects patients from exposure to non-autologous(allogeneic) blood, it conserves either autologous or allogeneic(banked, crossmatched) blood for its optimal use.

C. Limitations of Current Blood Substitutes

Attempts to produce blood substitutes (sometimes referred to as“oxygen-carrying plasma expanders”) have thus far produced products withmarginal efficacy or whose manufacture is tedious and expensive, orboth. Frequently, the cost of manufacturing such products is so highthat it effectively precludes the widespread use of the products,particularly in those markets where the greatest need exists (e.g.,emerging third-world economies).

The blood substitutes that have been developed previously are reviewedin various references (See e.g., Winslow, Robert M., “Hemoglobin-basedRed Cell Substitutes,” Johns Hopkins University Press, Baltimore[1992]). They can be grouped into the following three categories: i)perfluorocarbon-based emulsions, ii) liposome—encapsulated hemoglobin,and iii) modified cell-free hemoglobin. As discussed below, none hasbeen entirely successful, though products comprising modified cell-freehemoglobin are thought to be the most promising. Perfluorochemical-basedcompositions dissolve oxygen as opposed to binding it as a chelate. Inorder to be used in biological systems, the perfluorochemical must beemulsified with a lipid, typically egg-yolk phospholipid. Though theperfluorocarbon emulsions are inexpensive to manufacture, they do notcarry sufficient oxygen at clinically tolerated doses to be effective.Conversely, while liposome-encapsulated hemoglobin has been shown to beeffective, it is far too costly for widespread use (See e.g., Winslow,supra).

Most of the blood substitute products in clinical trials today are basedon modified hemoglobin. These products, frequently referred to ashemoglobin-based oxygen carriers (HBOCs), generally comprise ahomogeneous aqueous solution of a chemically-modified hemoglobin,essentially free from other red cell residues (stroma). Althoughstroma-free human hemoglobin is the most common raw material forpreparing a HBOC, other sources of hemoglobin have also been used. Forexample, hemoglobin can be obtained or derived from animal blood (e.g.,bovine hemoglobin) or from bacteria or yeast or transgenic animalsmolecularly altered to produce a desired hemoglobin product. (Seegenerally, Winslow, supra).

The chemical modification is generally one of intramolecularcrosslinking and/or oligomerization to modify the hemoglobin such thatits persistence in the circulation is prolonged relative to that ofunmodified hemoglobin, and its oxygen binding properties are similar tothose of blood. Intramolecular crosslinking chemically binds togethersubunits of the tetrameric hemoglobin unit to prevent the formation ofdimers which, as previously indicated, are prematurely excreted. (See,e.g., U.S. Pat. No. 5,296,465 to Rausch et al., hereby incorporated byreference).

The high costs of manufacturing HBOC products have greatly limited theircommercial viability. In addition, the present inventors have found thatknown HBOCs have a tendency to release excessive amounts of oxygen tothe tissues at the arteriole walls rather than the capillaries; this canresult in insufficient oxygen available for delivery by the HBOC to thetissues surrounding the capillaries. This is despite the fact that theinitial loading of the HBOC with oxygen may be relatively high, evenhigher than that normally achieved with natural red blood cells.

What is needed is a blood product that is relatively inexpensive tomanufacture and that delivers adequate amounts of oxygen to the tissues.

SUMMARY OF THE INVENTION

The present invention is directed at compositions comprising mixtures ofan oxygen-carrying component and a non-oxygen carrying component andmethods for their use. The compositions overcome the limited oxygendelivery characteristics of previous blood substitutes, and thereforelower doses may be used. They are a safer and more effective alternativeto currently available blood substitutes.

The present invention contemplates a means of improving the oxygendelivering capacity of an oxygen carrier by combining that carrier witha non-oxygen-carrying component like a conventional plasma expander. Inpreferred embodiments, the oxygen carrier (i.e., the oxygen-carryingcomponent) is a hemoglobin-based oxygen carrier. The hemoglobin may beeither native (unmodified); subsequently modified by a chemical reactionsuch as cross-linking, polymerization, or the addition of chemicalgroups (i.e., polyethyleneglycol, polyoxyethylene, or other adducts); orit may be recombinant or encapsulated in a liposome. Anon-oxygen-carrying plasma expander is any substance used for temporaryreplacement of red cells which has oncotic pressure (e.g., starches suchas hetastarch or pentastarch, dextran such as dextran-70 or dextran-90,albumin, or any other colloidal intravenous solution).

More specifically, it is contemplated that the compositions of thepresent invention will contain one or more of the following properties:i) viscosity at least half that of blood, ii) oncotic pressure higherthan that of plasma; iii) hemoglobin oxygen affinity higher than orequal to (i.e., P50 equal to or lower than) that of blood; and iv)oxygen capacity less than that of blood. It is not intended that theinvention be limited to how the compositions are used. A variety of usesare contemplated for the compositions of the present invention,including, but not limited to, the treatment of hemorrhage or use inhemodilution.

Particular non-oxygen carrying plasma expanders have been used (e.g.,for hemodilution) for a number of years, and their physiological effectsfollowing administration are well characterized. Previously, researchershave assumed that administration of an oxygen-carrying blood product(e.g., a blood substitute like an HBOC), should result in physiologicalcardiovascular responses similar to those observed followingadministration of non-oxygen carrying diluent materials of similarmolecular weight (e.g., dextran 70,000 MW, albumins and starches).Furthermore, researchers in the field of blood substitutes have beenworking under several other key assumptions. More specifically, prior tothe present invention, it has been thought that blood substitutes shouldhave viscosity less than that of blood, oxygen affinity similar to orequal to or lower than that of red cells, minimal colloidal osmotic(oncotic) pressure, and hemoglobin concentration as high as possible. Asdescribed in detail below, the compositions and methods of the presentinvention are counter-intuitive to some of these assumptions.

The present invention contemplates a blood product solution, comprisingan oxygen-carrying component and a non-oxygen carrying component, theblood product solution having oncotic pressure higher than that ofplasma and viscosity at least half that of blood. In some embodiments,the blood product solution further comprises oxygen affinity equal to orgreater than that of blood. In other embodiments, the blood productsolution further comprises oxygen capacity less than that of blood. Inparticular embodiments, the oxygen-carrying component is a polyethyleneglycol-modified hemoglobin. Furthermore, in certain embodiments thenon-oxygen-carrying component is a colloid starch. When thenon-oxygen-carrying component is a colloid starch, it has an averagemolecular weight of from approximately 200,000 daltons to approximately400,000 daltons is some embodiments. In particular embodiments, thecolloid starch is pentastarch.

The present invention also contemplates a blood product solution,comprising a) an oxygen-carrying component, the oxygen-carryingcomponent comprising a polyethylene glycol-modified hemoglobin; and b) anon-oxygen carrying component, the non-oxygen-carrying componentcomprising a colloid starch having an average molecular weight of fromapproximately 200,000 daltons to approximately 400,000 daltons. In someembodiments, the polyethylene glycol-modified hemoglobin compriseshemoglobin selected from the group consisting of animal hemoglobin,human hemoglobin, and recombinant hemoglobin. In particular embodiments,the colloid starch has an average molecular weight of from approximately225,000 daltons to approximately 300,000 daltons, and in otherembodiments the colloid starch is pentastarch. In still otherembodiments, the pentastarch comprises from approximately 20 percent toapproximately 80 percent by volume of the blood product solution,whereas the pentastarch comprises from approximately 40 percent toapproximately 60 percent by volume of the blood product in otherembodiments. Moreover, the blood product solution has a viscosity fromapproximately 2 centipoise to approximately 4.5 centipoise in particularembodiments.

The present invention also contemplates a method of enhancing oxygendelivery to the tissues of a mammal, comprising a) providing a bloodproduct solution, comprising an oxygen-carrying component and anon-oxygen carrying component, the blood product solution having oncoticpressure higher than that of plasma and viscosity at least half that ofblood; and b) administering the blood product solution to the mammal,thereby enhancing oxygen delivery to the tissues of the mammal. In someembodiments, the blood product solution further comprises oxygenaffinity equal to or greater than that of blood, while in otherembodiments the blood product solution further comprises oxygen capacityless than that of blood. In some embodiments, the oxygen-carryingcomponent is a polyethylene glycol-modified hemoglobin. Thenon-oxygen-carrying component is a colloid starch in particularembodiments; in some embodiments, the colloid starch has an averagemolecular weight of from approximately 200,000 daltons to approximately400,000 daltons. The colloid starch is pentastarch in still furtherembodiments.

In addition, the present invention contemplates a method of enhancingoxygen delivery to the tissues of a mammal, comprising a) providing ablood product solution, comprising i) an oxygen-carrying component, theoxygen-carrying component comprising a polyethylene glycol-modifiedhemoglobin, and ii) a non-oxygen carrying component, the non-oxygencarrying component comprising a colloid starch having an averagemolecular weight of from approximately 200,000 daltons to approximately350,000 daltons; and b) administering the blood product solution to themammal, thereby enhancing oxygen delivery to the tissues of the mammal.

In some embodiments, the polyethylene glycol-modified hemoglobincomprises hemoglobin selected from the group consisting of animalhemoglobin, human hemoglobin, and recombinant hemoglobin. In otherembodiments, the colloid starch has an average molecular weight of fromapproximately 200,000 daltons to approximately 400,000 daltons. In stillother embodiments, the colloid starch is pentastarch. In particularembodiments, the pentastarch comprises from approximately 20 percent toapproximately 80 percent by volume of the blood product.

In certain embodiments, the blood product solution has a viscosity offrom approximately 2 centipoise to approximately 4.5 centipoise.Finally, in other embodiments, the mammal is a human.

The present invention also provides an aqueous cell-free compositioncomprising hemoglobin, in which the hemoglobin is present in aconcentration of between 0.1 and 4.0 g/dl, and the aqueous compositionhas a viscosity that is greater than 2.5 cP. In some preferredembodiments, the viscosity of the aqueous composition is between 2.5 and4 cP. Thus, it is not intended that the present invention be limited toany viscosity that is greater than approximately 2.5 cP. Indeed, it iscontemplated that the present invention encompass compositions in whichthe viscosity is 6 cP or greater. In addition, the present inventionencompasses compositions in which the hemoglobin concentration is lessthan 0.1 or greater than 4 g/dl, although in particularly preferredembodiments, the hemoglobin concentration is between 0.1 and 4 g/dl.Furthermore, in some embodiments, the K* of the composition isapproximately equal or similar to that of a red blood cell suspensionwhen measured at the same hemoglobin concentration.

In other embodiments of the composition, the hemoglobin has an increasedaffinity for molecular oxygen as compared to red blood cells. Thepresent invention provides compositions that are suitable for use in anyanimal, including humans. Thus, in some embodiments, the hemoglobin ofthe composition has an increased affinity as compared to mammalian redblood cells, although in other embodiments, it is contemplated that thered blood cells are from reptiles, avians, or any other animal. In mostpreferred embodiments, the red blood cells used in this comparison arehuman red blood cells. In preferred embodiments, the composition has aP50 of less than 28 mm Hg. However, it is not intended that the presentinvention be limited to this P50 value, as in some embodiments, the P50is higher than 28 mm Hg.

In other embodiments, the composition further comprises a diluentselected from the group consisting of proteins, glycoproteins,polysaccharides, and other colloids. It is not intended that theseembodiments be limited to any particular diluent. Thus, it is intendedthat the diluent encompass solutions of albumin, other colloids, orother non-oxygen carrying components. In preferred embodiments, thediluent comprises polysaccharide. In other preferred embodiments, thepolysaccharide comprises starch. In particularly preferred embodiments,the starch comprises pentastarch.

In other embodiments, the hemoglobin within the composition issurface-modified. It not intended that these embodiments be limited toany particular type of surface modification. In preferred embodiments,the surface modification includes the use of polyalkylene oxide groupsof varying chain lengths and charges. In preferred embodiments, thehemoglobin is surface-modified with polyethylene glycol of varying chainlengths and charges. It is not intended that the surface modification belimited to any particular type or a single type of modification. It iscontemplated, that multiple types of surface-modifications will be madeto hemoglobin of the composition.

The present invention also provides an aqueous cell-free compositioncomprising surfaced-modified hemoglobin, wherein the surface-modifiedhemoglobin is present in a concentration of between 0.1 and 4.0 g/dl,and the aqueous composition has a viscosity that is greater than 2.5 cP.As discussed above, in some preferred embodiments, the viscosity of theaqueous composition is between 2.5 and 4 cP. Thus, it is not intendedthat the present invention be limited to any viscosity that is greaterthan approximately 2.5 cP. Indeed, it is contemplated that the presentinvention encompass compositions in which the viscosity is 6 cP orgreater. In further embodiments, the hemoglobin concentration is lessthan 0.1 or greater than 4 g/dl, although in particularly preferredembodiments, the hemoglobin concentration is between 0.1 and 4 g/dl. Insome embodiments, the K* of the composition is approximately equal orsimilar to that of a red blood cell suspension when measured at the samehemoglobin concentration.

In preferred embodiments of this composition, the hemoglobin has anincreased affinity for molecular oxygen as compared to red blood cells.As above, these embodiments are suitable for use in any animal,including humans. Thus, in some embodiments, the hemoglobin has anincreased affinity as compared to mammalian red blood cells, although inother embodiments, it is contemplated that the red blood cells are fromreptiles, avians, or any other animal. In most preferred embodiments,the red blood cells used in this comparison are human red blood cells.In other preferred embodiments, the composition has a P50 of less than28 mm Hg. However, it is not intended that the present invention belimited to this P50 value, as in some embodiments, the P50 is higherthan 28 mm Hg.

Furthermore, in other embodiments, the present invention providescompositions which further comprise a diluent selected from the groupconsisting of proteins, glycoproteins, polysaccharides, and othercolloids. It is not intended that the these embodiments be limited toany particular diluent. Thus, it is intended that the diluent encompasssolutions of albumin, other colloids, or other non-oxygen carryingcomponents. In preferred embodiments, the diluent comprisespolysaccharide. In other preferred embodiments, the polysaccharidecomprises starch. In particularly preferred embodiments, the starchcomprises pentastarch. In these embodiments, it not intended that thepresent invention be limited to any particular type of surfacemodification. In preferred embodiments, the surface modificationincludes the use of polyalkylene oxide groups of varying chain lengthsand charge. In preferred embodiments, the hemoglobin is surface-modifiedwith polyethylene glycol of varying chain lengths and charges.

The present invention further provides an aqueous cell-free compositioncomprising a mixture of hemoglobin and a diluent, wherein the hemoglobinis present in a concentration between 0.1 and 4 g/dl, and wherein thediluent is selected from the group consisting of proteins,glycoproteins, polysaccharides, and other colloids, and wherein theaqueous composition has a viscosity of at least 2.5 cP. As discussedabove, in some preferred embodiments, the viscosity of the aqueouscomposition is between 2.5 and 4 cP. Thus, it is not intended that theseembodiments be limited to any viscosity that is greater thanapproximately 2.5 cP. Indeed, it is contemplated that the presentinvention encompass compositions in which the viscosity is 6 cP orgreater. Furthermore, in some embodiments, the diluent comprises apolysaccharide, while in preferred embodiments, the diluent comprisesstarch, and in particularly preferred embodiments, the diluent comprisespentastarch. In addition, the present invention encompasses compositionsin which the hemoglobin concentration is less than 0.1 or greater than 4g/dl, although in particularly preferred embodiments, the hemoglobinconcentration is between 0.1 and 4 g/dl. In some embodiments, the K* ofthe composition is approximately equal or similar to that of a red bloodcell suspension when measured at the same hemoglobin concentration.

In some embodiments, the compositions comprise hemoglobin with anincreased affinity for molecular oxygen as compared to red blood cells.The present invention provides compositions that are suitable for use inany animal, including humans. Thus, in some embodiments, hemoglobin hasan increased affinity as compared to mammalian red blood cells, althoughin other embodiments, it is contemplated that the red blood cells arefrom reptiles, avians, or any other animal. In most preferredembodiments, the red blood cells used in this comparison are human redblood cells. In preferred embodiments, the composition has a P50 of lessthan 28 mm Hg. However, it is not intended that the present invention belimited to this P50 value, as in some embodiments, the P50 is higher.

As indicated above, these embodiments may also comprise hemoglobin thatis surface-modified. It not intended that the present invention belimited to any particular type of surface modification. In preferredembodiments, the surface modification includes the use of polyalkyleneoxide groups of varying chain lengths and charge. In preferredembodiments, the hemoglobin is surface-modified with polyethylene glycolof varying chain lengths and charges.

The present invention also provides methods comprising providing ananimal and an aqueous cell-free composition comprising hemoglobin,wherein the hemoglobin is present in a concentration of between 0.1 and4.0 g/dl, and the aqueous composition has a viscosity that is greaterthan 2.5 cP; and administering the aqueous composition to the animal. Inpreferred embodiments, the animal is a mammal, while in particularlypreferred embodiments, the animal is human. In some embodiments, thehuman is suffering from the symptoms of disease, pathology,insufficiency, or abnormality. In some embodiments, the human hassymptoms of disease, wherein the disease is selected from the groupconsisting of hypovolemic shock symptoms, hypoxia, chronic lung disease,ischemia, stroke, trauma, hemodilution, cardioplegia, cancer, anemia,sickle-cell anemia, septic shock, or disseminated intravascularcoagulation. However, it is not intended that the methods of the presentinvention be limited to the administration of the aqueous composition toalleviate any particular disease, condition, pathology, insufficiency,or abnormality. Rather, it is intended that the methods encompass anyand all applications for which the methods are suitable.

As above, the methods of present invention encompass an aqueouscell-free composition comprising hemoglobin, wherein the hemoglobin ispresent in a concentration of between 0.1 and 4.0 g/dl, and the aqueouscomposition has a viscosity that is greater than 2.5 cP. In somepreferred embodiments, the viscosity of the aqueous composition isbetween 2.5 and 4 cP. Thus, it is not intended that the presentinvention be limited to any viscosity that is greater than approximately2.5 cP. Indeed, it is contemplated that the present invention encompasscompositions in which the viscosity is 6 cP or greater. In addition, thepresent invention encompasses compositions in which the hemoglobinconcentration is less than 0.1 or greater than 4 g/dl, although inparticularly preferred embodiments, the hemoglobin concentration isbetween 0.1 and 4 g/dl. In some embodiments, the K* of the compositionis approximately equal or similar to that of a red blood cell suspensionwhen measured at the same hemoglobin concentration.

In alternative embodiments, the compositions comprise hemoglobin with anincreased affinity for molecular oxygen as compared to red blood cells.In addition, these embodiments are suitable for use with any animal,including humans. Thus, in some embodiments, hemoglobin has an increasedaffinity as compared to mammalian red blood cells, although in otherembodiments, it is contemplated that the red blood cells are fromreptiles, avians, or any other animal. In most preferred embodiments,the red blood cells used in this comparison are human red blood cells.In preferred embodiments, the composition has a P50 of less than 28 mmHg. However, it is not intended that the present invention be limited tothis P50 value, as in some embodiments, the P50 is higher than 28 mm Hg.

The other embodiments, the compositions which further comprise a diluentselected from the group consisting of proteins, glycoproteins,polysaccharides, and other colloids. It is not intended that the presentinvention be limited to any particular diluent. Thus, it is intendedthat the diluent encompass solutions of albumin, other colloids, orother non-oxygen carrying components. In preferred embodiments, thediluent comprises polysaccharide. In other preferred embodiments, thepolysaccharide comprises starch. In particularly preferred embodiments,the starch comprises pentastarch.

In yet other embodiments, the hemoglobin within the composition issurface-modified. It not intended that the present invention be limitedto any particular type of surface modification. In preferredembodiments, the surface modification includes the use of polyalkyleneoxide groups of varying chain lengths and charge. In preferredembodiments, the hemoglobin is surface-modified with polyethylene glycolof varying chain lengths and charges.

The present invention also provides methods comprising the steps ofproviding: an organ from an animal, and an aqueous cell-free compositioncomprising hemoglobin, wherein the hemoglobin is present in aconcentration of between 0.1 and 4.0 g/dl, and the aqueous compositionhas a viscosity that is greater than 2.5 cP; and perfusing the organwith said aqueous composition. In preferred embodiments, the animal is amammal, while in particularly preferred embodiments, the animal is ahuman. However, it is not intended that the methods be limited to humansor mammals. In preferred embodiments, the organ is selected from thegroup consisting of kidneys, liver, spleen, heart, pancreas, lung, andmuscle, although it is not intended that the methods of the present belimited to these organs, as any organ may be perfused with the aqueoussolution of the present invention.

In some preferred embodiments of the methods, the viscosity of theaqueous composition is between 2.5 and 4 cP. Thus, it is not intendedthat the present invention be limited to any viscosity that is greaterthan approximately 2.5 cP. Indeed, it is contemplated that the presentinvention encompass compositions in which the viscosity is 6 cP orgreater. In addition, the present invention encompasses compositions inwhich the hemoglobin concentration is less than 0.1 or greater than 4g/dl, although in particularly preferred embodiments, the hemoglobinconcentration is between 0.1 and 4 g/dl. In some embodiments, the K* ofthe composition is approximately equal or similar to that of a red bloodcell suspension when measured at the same hemoglobin concentration.

These embodiments also provide compositions comprising hemoglobin withan increased affinity for molecular oxygen as compared to red bloodcells. As above, these embodiments are suitable for use in any animal,including humans. Thus, in some embodiments, hemoglobin has an increasedaffinity as compared to mammalian red blood cells, although in otherembodiments, it is contemplated that the red blood cells are fromreptiles, avians, or any other animal. In most preferred embodiments,the red blood cells used in this comparison are human red blood cells.In preferred embodiments, the composition has a P50 of less than 28 mmHg. However, it is not intended that the present invention be limited tothis P50 value, as in some embodiments, the P50 is higher than 28 mm Hg.

The other embodiments, the compositions further comprise a diluentselected from the group consisting of proteins, glycoproteins,polysaccharides, and other colloids. It is not intended that the presentinvention be limited to any particular diluent. Thus, it is intendedthat the diluent encompass solutions of albumin, other colloids, orother non-oxygen carrying components. In preferred embodiments, thediluent comprises polysaccharide. In other preferred embodiments, thepolysaccharide comprises starch. In particularly preferred embodiments,the starch comprises pentastarch.

In yet other embodiments, the hemoglobin within the composition issurface-modified. It not intended that the present invention be limitedto any particular type of surface modification. In preferredembodiments, the surface modification includes the use of polyalkyleneoxide groups of varying chain lengths and charge. In preferredembodiments, the hemoglobin is surface-modified with polyethylene glycolof varying chain lengths and charges.

It is not intended that the present invention be limited to anyparticular oncotic pressure. Indeed, it is intended that thecompositions of the present invention encompass a range of oncoticpressure. In some embodiments, the oncotic pressure ranges from 70 to 80mm Hg, while in the most preferred embodiments, the oncotic pressure isapproximately 90 mm Hg. However, in other embodiments, the oncoticpressure can be as low as 60 mm Hg. Furthermore, it is intended that thepresent invention encompass hypooncotic, hyperoncotic, and isooncoticpressures. As used herein, the term “hyperoncotic” encompasses anyoncotic pressure that is greater than 25 mm Hg, although in preferredembodiments, solutions with oncotic pressures of 20-60 mm Hg areprovided. In some embodiments of the methods of the present invention,it is contemplated that the composition chosen for administration willbe customized to the particular needs of the animal. The presentinvention provides the means to customize the composition to meet theneeds of various clinical and veterinary uses.

FIG. 19 provides a graph showing the hemoglobin concentration andviscosity of various hemoglobin preparations. The square positionedwithin this graph (i.e., at approximately 2.5-4 cP and 0.1 to 4 g/dlhemoglobin) indicates the properties of the most preferred compositionsof the present invention. As indicated, the only hemoglobin solutionthat meets the criteria is the “Hemospan” solution which was madeaccording to the methods of the present invention. The other samples inthis graph include blood, PEG-Hb (Enzon), PHP (Apex), and αα-hemoglobin(US Army). As discussed in more detail below, the characteristics of thecompositions of the present invention provide many heretofore unknownand unexpected advantages.

The present invention further provides a method comprising: providing i)liganded hemoglobin, ii) means for treating hemoglobin, and iii) meansfor surface decorating hemoglobin; treating the liganded hemoglobin withthe treating means under conditions such that a treated hemoglobin isproduced having greater affinity for molecular oxygen than unligandedhemoglobin; and surface decorating the treated hemoglobin with thesurface decorating means.

In some embodiments of the method, the means for treating is selectedfrom the group consisting of crosslinking means and polymerizing means.In alternative embodiments, the surface decoration of step (c) comprisesreacting said treated hemoglobin with a polyalkylene oxide.

The present invention also provides a method comprising: providing i)liganded hemoglobin, ii) means for treating hemoglobin selected from thegroup consisting of crosslinking means and polymerizing means, and iii)means for surface decorating hemoglobin; treating the ligandedhemoglobin with the treating means under conditions such that a treatedhemoglobin is produced having greater affinity for molecular oxygen thanunliganded hemoglobin; and surface decorating the treated hemoglobinwith the surface decorating means. In some embodiments of the method,the surface decoration of step (c) comprises reacting the treatedhemoglobin with a polyalkylene oxide.

The present invention further provides a method comprising: providing i)hemoglobin, ii) means for enzymatically treating hemoglobin (e.g., withenzymes such as carboxy peptidase), and iii) means for surfacedecorating hemoglobin; treating the liganded hemoglobin with theenzymatic treating means under conditions such that an enzymaticallytreated hemoglobin is produced having greater affinity for molecularoxygen than hemoglobin in red blood cells; and surface decorating theenzymatically treated hemoglobin with the surface decorating means.

Definitions

To facilitate understanding of the invention set forth in the disclosurethat follows, a number of terms are defined below.

The phrase “oxygen capacity less than that of blood” means that when theoxygen capacity of the blood product solutions of the present inventionis compared with that of blood, the oxygen capacity of the blood productsolutions is less. The oxygen capacity of the blood product solutions ofthe present invention is not required to be less than that of blood byany particular amount. Oxygen capacity is generally calculated fromhemoglobin concentration, since it is known that each gram of hemoglobinbinds 1.34 mL of oxygen. Thus, the hemoglobin concentration in g/dLmultiplied by the factor 1.34 yields the oxygen capacity in mL/dL. Thepresent invention contemplated the use of a suitable commerciallyavailable instruments to measure hemoglobin concentration, including theB-Hemoglobin Photometer (Hemocue, Inc.). Similarly, oxygen capacity canbe measured by the amount of oxygen released from a sample of hemoglobinor blood by using, for example, a fuel-cell instrument (e.g.,Lex-O₂-Con; Lexington Instruments).

The phrase “oxygen affinity equal to or greater than that of blood”means that when the oxygen affinity of the blood product solutions ofthe present invention is compared with that of blood, the oxygenaffinity of the blood product solutions is greater. The oxygen capacityof the blood product solutions of the present invention is not requiredto be greater than that of blood by any particular amount. The oxygenaffinity of whole blood (and components of whole blood such as red bloodcells and hemoglobin) can be measured by a variety of methods known inthe art. (See, e.g., Vandegriff and Shrager in Methods in Enzymology(Everse et al., eds.) 232:460 [1994]). In preferred embodiments, oxygenaffinity may be determined using a commercially available HEMOX®Analyzer (TCS Medical Products). (See, e.g., Winslow et al., J. Biol.Chem., 252(7):2331-37 [1977]).

The phrase “oncotic pressure higher than that of plasma” means that whenthe oncotic pressure of the blood product solutions of the presentinvention is compared with that of plasma, the oxygen affinity of theblood product solutions is greater. The oncotic pressure of the bloodproduct solutions of the present invention is not required to be greaterthan that of blood by any particular amount. Oncotic pressure may bemeasured by any suitable technique; in preferred embodiments, oncoticpressure is measured using a Colloid Osmometer (Wesco model 4420).

The phrase “viscosity at least half of that of blood” means that whenthe viscosity of the blood product solutions of the present invention iscompared with that of blood, the oxygen affinity of the blood productsolutions is at least 50% of that of blood; in addition, the viscositymay be greater than that of blood. Preferably, viscosity is measured at37° C. in a capillary viscometer using standard techniques. (SeeReinhart et al., J. Lab. Clin. Med. 104:921-31 [1984]). Moreover,viscosity can be measured using other methods, including a rotatingcone-and-plate viscometer such as those commercially available fromBrookfield. The viscosity of blood is approximately 4 centipoise. Thus,at least half of the blood value corresponds to at least approximately 2centipoise.

The term “blood product” refers broadly to formulations capable of beingintroduced into the circulatory system of the body and carrying andsupplying oxygen to the tissues. While the term “blood products”includes conventional formulations (e.g., formulations containing thefluid and/or associated cellular elements and the like that normallypass through the body's circulatory system, including, but not limitedto, platelet mixtures, serum, and plasma), the preferred blood productsof the present invention are “blood product mixtures.” As used herein,blood product mixtures comprise a non-oxygen-carrying component and anoxygen-carrying component.

The term “oxygen-carrying component” refers broadly to a substancecapable of carrying oxygen in the body's circulatory system anddelivering at least a portion of that oxygen to the tissues. Inpreferred embodiments, the oxygen-carrying component is native ormodified hemoglobin. As used herein, the term “hemoglobin” refers to therespiratory protein generally found in erythrocytes that is capable ofcarrying oxygen. Modified hemoglobin includes, but is not limited to,hemoglobin altered by a chemical reaction such as cross-linking,polymerization, or the addition of chemical groups (e.g.,polyethyleneglycol, polyoxyethylene, or other adducts). Similarly,modified hemoglobin includes hemoglobin that is encapsulated in aliposome.

The present invention is not limited by the source of the hemoglobin.For example, the hemoglobin may be derived from animals and humans;preferred sources of hemoglobin are cows and humans. In addition,hemoglobin may be produced by other methods, including recombinanttechniques. A most preferred oxygen-carrying-component of the presentinvention is “polyethylene glycol-modified hemoglobin.”

The term “polyethylene glycol-modified hemoglobin” refers to hemoglobinthat has been modified such that it is associated with polyethyleneglycol (α-Hydro-ω-hydroxypoly-(oxy-1,2-ethanediyl); generally speaking,the modification entails covalent binding of polyethylene glycol (PEG)to the hemoglobin. PEGs are liquid and solid polymers of the generalchemical formula H(OCH₂CH₂)_(n)OH, where n is greater than or equal to4. PEG formulations are usually followed by a number that corresponds toits average molecular weight; for example, PEG-200 has a molecularweight of 200 and a molecular weight range of 190-210. PEGs arecommercially available in a number of formulations (e.g., Carbowax,Poly-G, and Solbase).

The term “non-oxygen-carrying component” refers broadly to substanceslike plasma expanders that can be administered, e.g., for temporaryreplacement of red blood cell loss. In preferred embodiments of theinvention, the non-oxygen-carrying component is a colloid (i.e., asubstance containing molecules in a finely divided state dispersed in agaseous, liquid, or solid medium) which has oncotic pressure (colloidosmotic pressure prevents, e.g., the fluid of the plasma from leakingout of the capillaries into the interstitial fluid). Examples ofcolloids include hetastarch, pentastarch, dextran-70, dextran-90, andalbumin.

Preferred colloids of the present invention include starches likehetastarch and pentastarch. Pentastarch (hydroxyethyl starch) is thepreferred colloid starch of the present invention. Pentastarch is anartificial colloid derived from a starch composed almost entirely ofamylopectin. Its molar substitution is 0.45 (i.e., there are 45hydroxyethyl groups for every 100 glucose units); hydroxyethyl groupsare attached by an ether linkage primarily at C-2 of the glucose unit(and less frequently at C-3 and C-6). The polymerized glucose units ofpentastarch are generally connected by 1-4 linkages (and less frequentlyby 1-6 linkages), while the degree of branching is approximately 1:20(i.e., there is one branch for every 20 glucose monomer units). Theweight average molecular weight of pentastarch is about 250,000 with arange of about 150,000 to 350,000. Unless otherwise indicated, referenceto the “average molecular weight” of a substance refers to the weightaverage molecular weight. Pentastarch is commercially available (e.g.,DuPont Merck) as a 10% solution (i.e., 10 g/100 mL); unless otherwiseindicated, reference to blood product solutions comprising pentastarch(and other non-oxygen-carrying components as well as oxygen-carryingcomponents) is on a volume basis.

The phrase “enhancing oxygen delivery to the tissues of a mammal” refersto the ability of a fluid (e.g., a blood product) introduced into thecirculatory system to deliver more oxygen to the tissues than would bedelivered without introduction of the fluid. To illustrate, a patientmay experience substantial blood loss following acute hemorrhage,resulting in decreased transport of oxygen to the tissues via the blood.The administration of a blood product to the patient can supplement theability of the patient's own blood to deliver oxygen.

The term “mixture” refers to a mingling together of two or moresubstances without the occurrence of a reaction by which they would losetheir individual properties. The term “solution” refers to a liquidmixture. The term “aqueous solution” refers to a solution that containssome water. In many instances, water serves as the diluent for solidsubstances to create a solution containing those substances. In otherinstances, solid substances are merely carried in the aqueous solution(i.e., they are not dissolved therein). The term aqueous solution alsorefers to the combination of one or more other liquid substances withwater to form a multi-component solution.

The term “approximately” refers to the actual value being within a rangeof the indicated value. In general, the actual value will be between 10%(plus or minus) of the indicated value.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B are a diagrammatic cross-sectional illustration of the flowof whole blood (FIG. 1A) and a hemoglobin-based oxygen carrier (FIG. 1B)through an arterial vessel.

FIG. 2 depicts a plot of flow velocity in the microcirculation as afunction of hematocrit reductions with dextran hemodilution and salinehemodilution.

FIG. 3 graphically presents mean arterial blood pressure in rats priorto and during an exchange transfusion (arrow) with Hemolink® (▾),pentastarch (▴) and a 50/50 (volume/volume) mixture ofHemolink®+pentastarch (∇).

FIG. 4 graphically presents mean arterial blood pressure in ratsfollowing exchange transfusion with Hemolink® (▾), pentastarch (▴) and a50/50 (volume/volume) mixture of Hemolink®+pentastarch (∇), during a 60%blood volume hemorrhage.

FIG. 5 depicts rat survival following exchange transfusion withpentastarch (▴), αα-Hb (▪), PEG-Hb (●), pentastarch+αα-Hb (□), andpentastarch+PEG-Hb (∘) and after the initiation of a 60% hemorrhage.

FIG. 6A-D graphically depict the acid-base status of control rats (♦)and of rats following exchange transfusion with pentastarch (▴), αα-Hb(▪), PEG-Hb (●), pentastarch+αα-Hb (□), and pentastarch+PEG-Hb (∘) andafter the initiation of a 60% hemorrhage. FIG. 6A depicts PaO₂, FIG. 6Bdepicts PaCO₂, FIG. 6C depicts arterial pH, and FIG. 6D depicts baseexcess.

FIG. 7 graphically depicts the production of lactic acid in control rats(♦) and of rats following exchange transfusion with pentastarch (▴),αα-Hb (▪), PEG-Hb (●), pentastarch+αα-Hb (□), and pentastarch+PEG-Hb (∘)and after the initiation of a 60% hemorrhage.

FIG. 8A depicts mean arterial blood pressure in control rats (♦) and ofrats following exchange transfusion with pentastarch (▴), PEG-Hb (●),and Pentaspan+PEG-Hb (∘) at time −30 minutes, and after the initiationof a 60% hemorrhage at time 0 minutes.

FIG. 8B depicts mean arterial blood pressure in control rats (♦), andrats following exchange transfusion with pentastarch (▴, point B), αα-Hb(▪, point B), and pentastarch+αα-Hb (□, point A), and after theinitiation of a 60% hemorrhage (point C).

FIG. 9 depicts cardiac output in control rats (♦) and in rats followingexchange transfusion with pentastarch (▴), αα-Hb (▪), PEG-Hb (●), andpentastarch+PEG-Hb (∘) and after the initiation of a 60% hemorrhage at 0minutes.

FIG. 10 depicts systemic vascular resistance in control rats (♦) and ofrats following exchange transfusion with pentastarch (▴), αα-Hb (▪),PEG-Hb (●), and pentastarch+PEG-Hb (∘) and after the initiation of a 60%hemorrhage at 0 minutes.

FIG. 11 depicts animal survival following exchange transfusion withpentastarch (▴), αα-Hb (▪), and pentastarch+αα-Hb (□) after theinitiation of a 60% hemorrhage.

FIG. 12 depicts animal survival following exchange transfusion withhetastarch (x), Hemolink® (▾), Hemolink®+pentastarch (∇), andhetastarch+Hemolink® (⋄) and after the initiation of a 60% hemorrhage.

FIG. 13 provides an illustration of a Krogh cylinder.

FIG. 14 provides a schematic of a capillary system.

FIG. 15. is a graph showing the exit PO₂ compared to the residence timeof red blood cells, A₀ hemoglobin, αα-hemoglobin, and PEG-Hb.

FIG. 16. is a graph showing the saturation compared with the residencetime of red blood cells, A₀ hemoglobin, αα-hemoglobin, and PEG-Hb.

FIG. 17 is a graph showing the K* compared to the residence time of redblood cells, A₀ hemoglobin, αα-hemoglobin, and PEG-Hb.

FIG. 18 is a graph showing the MAP over time for ₀ hemoglobin,αα-hemoglobin, and PEG-Hb.

FIG. 19 is a graph showing the hemoglobin concentration and viscosity ofvarious hemoglobin solutions.

DESCRIPTION OF THE INVENTION

The present invention relates generally to blood products, and moreparticularly to compositions comprising a mixture of an oxygen-carryingcomponent and a non-oxygen-carrying component and methods for the usethereof. The compositions and methods of the present invention result inimproved oxygen delivering capacity of hemoglobin-based oxygen carriers.Generally speaking, the compositions of the present invention willexhibit one or more of the following properties: i) viscosity at leasthalf that of blood; ii) oncotic pressure higher than that of plasma;iii) hemoglobin oxygen affinity higher than or equal to (i.e., P50 equalto or lower than) that of blood; and iv) oxygen capacity less than thatof blood. Because of the more efficient utilization of the oxygencarried by the HBOC in terms of tissue oxygenation, the compositions ofthe present invention comprise a substantially reduced hemoglobincontent and are generally less expensive to formulate.

The description of the invention is divided into: I) The Nature ofOxygen Delivery and Consumption; II) Facilitated Diffusion and TheDesign of Hemoglobin-Based Oxygen Carriers; III) Clinical and OtherApplications of the Present Invention; IV) The Oxygen-carrying Componentof the Blood Products of the Present Invention; V) The Non-oxygenCarrying Component of the Blood Products of the Present Invention; andVI) Blood Product Compositions. Each section will be discussed in turnbelow.

I. The Nature of Oxygen Delivery and Consumption

Although the successful use of the compositions and methods of thepresent invention do not require comprehension of the underlyingmechanisms of oxygen delivery and consumption, basic knowledge regardingsome of these putative mechanisms may assist in understanding thediscussion that follows. As previously indicated, it has generally beenassumed that the capillaries are the primary conveyors of oxygen to thetissue; however, regarding tissue at rest, current findings indicatethat there is approximately an equipartition between arteriolar andcapillary oxygen release. That is, hemoglobin in the arterial system isbelieved to deliver approximately one-third of its oxygen content in thearteriolar network and one-third in the capillaries, while the remainderexits the microcirculation via the venous system. The arteriesthemselves comprise a site of oxygen utilization (e.g., the artery wallrequires energy to effect regulation of blood flow through contractionagainst vascular resistance). Thus, the arterial wall is normally asignificant site for the diffusion of oxygen out of the blood. However,current oxygen-delivering compositions (e.g., HBOCs) may release toomuch of their oxygen content in the arterial system, and thereby inducean autoregulatory reduction in capillary perfusion.

The rate of oxygen consumption by the vascular wall, i.e., thecombination of oxygen required for mechanical work and oxygen requiredfor biochemical synthesis, can be determined by measuring the gradientat the vessel wall. Present technology allows accurate oxygen partialpressure measurements in vessels on the order of 50 microns diameter.The measured gradient is directly proportional to the rate of oxygenutilization by the tissue in the region of the measurement. Suchmeasurements show that the vessel wall has a baseline oxygen utilizationwhich increases in inflammation and constriction, and is lowered byrelaxation.

The vessel wall gradient is inversely proportional to tissueoxygenation. Vasoconstriction increases the oxygen gradient (tissuemetabolism), while vasodilation lowers the gradient. Higher gradientsare indicative of the fact that more oxygen is used by the vessel wall,while less oxygen is available for the tissue. The same phenomenon isbelieved to be present throughout the microcirculation.

The present invention demonstrates that increased blood PO₂ (which canbe obtained, e.g., by hemodilution) through administration of aconventional oxygen-carrying solution (e.g., a HBOC), thoughsuperficially a beneficial outcome of the altered blood flowcharacteristics and blood oxygen carrying capacity of the resultingcirculatory fluid, carries with it significant disadvantages. That is,when the hemoglobin carrying the oxygen is evenly distributed in thevessel as opposed to being contained in RBCs, a different set of factorsinfluencing oxygen delivery apparently come into play. The presentinvention provides a means of alleviating these disadvantages, namely byproviding and using an aqueous solution of an oxygen-carrying component(e.g., modified hemoglobin) and a non-oxygen-carrying component (e.g., anon-proteinaceous colloid such as dextran or pentastarch). Among otherattributes, the compositions of the present invention can bemanufactured at a much lower cost than that of normal HBOCs and providea blood substitute of increased viscosity.

FIG. 1A diagrammatically illustrates, in cross section, an arteriolehaving a wall (2) surrounding the flow passage therethrough. The wall inturn, is surrounded by muscle (1). As previously indicated, normal wholeblood consists essentially of red blood cells (3) and plasma (4).Substantially all (approximately 97%) of oxygen carried by the blood isassociated with the hemoglobin and is inside the red blood cells (3);only about 3% of the oxygen is in the plasma component.

Accordingly, the oxygen availability to the artery wall (2) is limitedby the surface area of the RBCs and the rate of diffusion of oxygenthrough the RBC membrane and surrounding unstirred plasma. The arterywalls receive an amount of oxygen proportional to the spacing betweenRBCs and the mean distance for diffusion from RBCs to the wall.

For comparison purposes, FIG. 1B diagrammatically illustrates oxygendelivery when an artery is perfused with a HBOC (5) mixed with wholeblood. In this situation, the component of the HBOC that directly bindsoxygen is homogeneously distributed throughout the HBOC (5) and theoxygen is available for diffusion to all parts of the surface of theartery wall (2). Thus, oxygen availability to the artery wall (2) isgreatly increased, effectively causing an increase of PO₂ in thearterial system. Though the present invention does not require anunderstanding of the precise mechanisms, it is believed that arterialwall and muscle reactions (e.g., increased metabolism of the cellularcomponents of the vessel wall as a consequence of energy-consumingvasoconstrictor effects) take place in an attempt to maintain the PO₂ ofthe tissue; this is evidenced by the establishment of a large gradientof oxygen partial pressure across the arterial wall aimed at maintainingarteriolar partial oxygen pressure constant. As a result, there isexcessive loss of oxygen from the blood-HBOC mixture at the arterialwalls, and, concomitantly, insufficient oxygen is available forcapillary delivery to the tissues.

Though a precise understanding of the underlying mechanism is notrequired in order to practice the present invention, the presentinvention is based upon the discovery that a HBOC tends to release toomuch of the oxygen it carries at the artery walls, resulting in reactionof the arterial walls to the excess oxygen and oxygen deficiency at thecapillaries. As alluded to above, researchers have previously assumedthat administration of a blood substitute (e.g., a HBOC) should resultin physiological cardiovascular responses similar to those observed uponadministration of non-oxygen carrying diluent materials of similarmolecular weight. However, it has been observed that HBOCs causephysiological reactions that differ from those found withnon-oxygen-carrying plasma expanders. The dilution of RBCs, accompaniedby the maintenance of intrinsic oxygen delivering capacity of thecomposition (i.e., because the blood substitute composition is itself anoxygen carrier), changes the distribution of oxygen in the circulatorysystem, increasing the PO₂ in the arteriolar segment. As discussedfurther below, this in turn appears to lead to the reaction of themuscles lining the arterial walls to the excess oxygen availability. Incontrast, the compositions of the present invention result in increasedoxygen delivery to the tissues surrounding the capillaries.

As set forth in the preceding discussion, the suitability of a bloodproduct should be determined by analysis of its systemic effects, andhow such effects, in conjunction with the altered transport propertiesof the circulating fluid, influence transport microcirculatory function.

II. Facilitated Diffusion and the Design of Hemoglobin-Based OxygenCarriers

Vasoconstriction is one of the most perplexing problems in thedevelopment of a safe and efficacious red cell substitute. When infusedinto animals and humans, many hemoglobin-based solutions producesignificant hypertension, increased vascular resistance and decreased O₂transport. This phenomenon has been observed in both the systemic andpulmonary circulations in models of clinical use (Hess et al., J. Appl.Physiol., 74: 1769-78 [1993], Keipert et al., Transfusion 33: 701-8[1993]) and in humans (Kasper et al., Biochem., 31: 7551-9 [1992]).

Vasoactivity is usually attributed to the avidity with which hemoglobincombines with nitric oxide, the endothelium-derived relaxing factor. TheNO affinity of model hemoglobins however does not correlate with theeffect on mean arterial blood pressure in rats (Rohlfs et al., In R. M.Winslow et al., (eds.), Advances in Blood Substitutes. IndustrialOpportunities and Medical Challenges, Birkhauser, Boston [1997], pp.298-327 [1997]), and it is possible that oversupply of O₂ due todiffusion of HbO₂ or removal of NO due to diffusion of HbNO also playsan additional, if not exclusive, role.

Increased rates of O₂ uptake and release by cell-free hemoglobincompared to red blood cells have been predicted (See e.g., Homer,Microvasc. Res., 22: 308-23 [1981]; Federspiel and Popel, Microvasc.Res., 32: 164-189 [1986]) and shown in vitro (Page et al., In R. M.Winslow et al., (eds.), Blood Substitutes. New Challenges, Birkhauser,Boston [1996], pp. 132-145). However, attempts to demonstrate augmentedtransport by O₂ diffusion in vivo by cell-free hemoglobin have beenunsuccessful (See, Biro, Can. J. Physiol. Pharmacol., 69: 1656-1662[1991]; Hogan et al., Adv. Exp. Med. Biol., 361: 375-378 [1994]; andHogan et al., J. Appl. Physiol., 361: 2470-5 [1992]). Although anunderstanding of the mechanism is not necessary in order to make and usethe present invention, during the development of the present invention,it was determined, shown, by measurements in artificial capillaries,that cell-free hemoglobin does, indeed, increase the availability of O₂to the surrounding medium.

In normal blood, O₂ moves from the red blood cell to the vessel wall bysimple diffusion. When hemoglobin is present in the plasma space, O₂ canalso move bound to hemoglobin as HbO₂. This second process is called“facilitated diffusion.” During the development of the presentinvention, properties of cell-free hemoglobin that modulate thisfacilitated diffusion were identified. Using this knowledge, hemoglobinsthat demonstrate diffusive O₂ transport similar to that of red bloodcells by reduced facilitated diffusion were prepared. It was alsoconfirmed that these example molecules do not produce vasoconstrictionin animals. Surprisingly, it was found that increased viscosity,increased O₂ affinity (reduced P50), and increased molecular size arethe key properties required for a cell-free hemoglobin to avoidvasoactivity and to enable success as a red cell substitute.

In addition, the present invention provides teachings regarding theoptimal properties of hemoglobin-based blood substitutes in regard tooxygen affinity, viscosity and molecular size and a method to evaluatesuch products by an instrument based on an artificial capillary. Thismethod enables the quantitative determination of the ability of a bloodsubstitute to transfer O₂ (or any other gas such as CO₂, NO, or CO)across a capillary membrane as a model of in vivo gas transfer.

A. Facilitated Diffusion

During the development of the present invention, it was shown thatunexpectedly, arterioles, particularly at the A2/A3 level consume largeamounts of O₂. This was determined by a technique for measuring O₂concentration in localized areas of the microcirculation (Torres andIntaglietta, Am. J. Physiol., 265: H1434-H1438 [1993]). These resultsindicate that these arterioles are capable of prodigious metabolicactivity. Innervation of these arterioles is particularly dense(Saltzman et al., Microvasc. Res., 44: 263-273 [1992]), suggesting thatthey regulate downstream capillary blood flow. Based on these results,increasing the O₂ available to these arterioles would be expected toprovide a potent stimulus to engage mechanisms that regulate thedelivery of O₂ to capillary beds (autoregulation). Although anunderstanding of the exact biochemical mechanism(s) which underlie theseevents is not necessary in order to use the present invention, it iscontemplated that they could be mediated by O₂— or NO— sensitivepathways; the presence of hemoglobin, free in the plasma space, as in a“blood substitute” is likely to engage these mechanisms because of itscapacity for facilitated diffusion.

The transport of O₂ in the blood by two pathways (O₂ and HbO₂ diffusion)can be expressed mathematically. The transport (flux, −J) of O₂ to thevessel wall is the sum of the diffusion of free (O₂) and chemicallybound oxygen (HbO₂): $\begin{matrix}{{- J} = {\frac{D_{O_{2}}\alpha\quad\Delta\quad P\quad O_{2}}{\Delta\quad X} + \frac{{D_{{HbO}_{2}}\lbrack{Hb}\rbrack}_{T}\Delta\quad Y}{\Delta\quad X}}} & (1)\end{matrix}$

where D₀₂ and D_(HbO2) are the diffusion constants for O₂ and cell-freeHbO₂, respectively, α is the solubility of O₂ in plasma, ΔPO₂ is thedifference in partial pressure of O₂ inside and outside the vessel, ΔYis the gradient of hemoglobin saturation from the center of the vesselto its wall, and [Hb]_(T) is the total cell-free hemoglobinconcentration. D₀₂ and D_(Hbo2) have been measured experimentally instatic solution (Table 1). The distance for diffusion, ΔX, is consideredto be the same for the two molecules, O₂ and HbO₂. The references citedin Table 1 are: Wittenberg, Physiol. Rev., 50(4): 559-636 [1970], andBouwer, Biochim. Biophys. Acta 1338: 127-136 [1977]). TABLE 1 Values ForDiffusion Constants From The Literature D_(O2) (cm²/sec) D_(HbO2)(cm²/sec) Wittenberg 2.13 × 10⁻⁵ 11.3 × 10⁻⁷ Bouwer 1.40 × 10⁻⁵  7.0 ×10⁻⁷ Mean 1.76 × 10⁻⁵ 9.15 × 10⁻⁷

Table 1 shows that D_(HbO2) is about 1/20^(th) of D₀₂. However becausethe solubility of O₂ in plasma is low (α=1.2074 μM/Torr), and D₀₂ isrelatively high, when plasma hemoglobin concentration is only 3 mM (4.83g/dl) at PO₂ of 100 Torr, the product of diffusion and concentration(the numerators in equation 1) for free O₂ and HbO₂ are nearly equal.Thus plasma hemoglobin contributes as much O₂ as dissolved O₂,effectively doubling the amount of O₂ available from red blood cells.These relationships are shown quantitatively in Table 2. TABLE 2 TheProduct Of Diffusion And Concentration For Dissolved O₂ vs. HbO₂Concentration at Diffusion constant Concentration × 100 Torr, mM (seetable 3) Diffusion O₂ 0.1207  176 × 10⁻⁷ 2.48 × 10⁻⁶ HbO₂ 3.0 9.15 ×10⁻⁷ 2.74 × 10⁻⁶

In order to develop a strategy to minimize the facilitated diffusion ofO₂ by plasma HbO₂, it was necessary to analyze the biophysicalproperties which contribute to it. Because water is much smaller thanHbO₂, D_(HBO2) is a function of viscosity and molecular radius, asdefined by the Stokes-Einstein equation: $\begin{matrix}{D_{{HbO}_{2}} = \frac{k\quad T}{6\quad\eta_{solution}r_{{HbO}_{2}}}} & (2)\end{matrix}$where k is Boltzman's constant, η_(solution) is the viscosity of thesolution, and r_(HbO2) is the radius of the hemoglobin molecule (HbO₂).For molecular oxygen, where the molecular radius (r_(O2)) isapproximately the same as that of water, the Stokes-Einstein equationbecomes: $\begin{matrix}{D_{O_{2}} = \frac{k\quad T}{4\quad\eta_{solution}r_{O_{2}}}} & (3)\end{matrix}$

Thus, for both HbO₂ and dissolved O₂ their diffusivities are inverselyrelated to the viscosity of the macromolecular solutions. For cell-freehemoglobin, hemoglobin molecular size is an additional factor in thatD_(HbO2) is inversely proportional to the molecular size of hemoglobin(r_(HbO2) in Equation 2). Thus this analysis predicts that two potentialstrategies to reduce or eliminate facilitated diffusion by cell-freehemoglobin is increasing the molecular radius of the molecule andincreasing solution viscosity.

Further analysis of the equation 1 leads to an understanding of anadditional strategy to defeat this mechanism. The gradient along whichHbO₂ diffuses is [Hb]_(T)ΔY and the distance through which HbO₂ mustdiffuse (ΔX_(HbO2)). The quantity ΔY at a given PO₂ is the slope of theoxygen equilibrium curve at that PO₂ and is dependent on the shape ofthe curve (a property of the hemoglobin molecule) and its position(i.e., P50).

To summarize, the total O₂ transferred in a cylindrical section of theKrogh cylinder (see FIG. 1) can be described as follows: $\begin{matrix}{{\Delta\quad O_{2_{r}}} = {( \frac{\pi\quad r^{2}}{R} )\lbrack {( \frac{D_{O_{2}}\alpha\quad\Delta\quad P\quad O_{2}}{\Delta\quad X_{O_{2}}} ) + ( \frac{{D_{{HbO}_{2}}\lbrack{Hb}\rbrack}_{T}\Delta\quad Y}{\Delta\quad X_{{HbO}_{2}}} )} \rbrack}} & (4)\end{matrix}$

In this equation, r is the radius of the capillary, and R is the flowrate. The equation shows the contribution of HbO₂ diffusion to total O₂transport. This form of the O₂ transfer equation has the interestingproperty in that it shows that the contribution of the HbO₂ diffusion isdependent on 4 variables: the diffusion constant (D_(HbO2)), hemoglobinconcentration ([Hb]_(T)), the difference in saturation between thecenter and the edge of the capillary (ΔY) and the distance for diffusionof HbO₂ (ΔX_(HBO2)).

Equation 4 reveals a number of strategies that can be employedindependently or in combination to modulate O₂ flux (ΔO_(2T)). Thestrategies are defined by the relationship of ΔO_(2T) to the alterablesolution properties such that ΔO_(2T) is:

-   -   (1) inversely proportional to solution viscosity (η), according        to Eqs. 2 and 3, through changes in both DO₂ and DHbO₂;    -   (2) inversely proportional molecular size (r_(HbO2)), according        to Eq. 2, through a change D_(HbO2;)    -   (3) directly proportional to [Hb]T; and    -   (4) directly proportional to ΔY (ΔY can be altered by changing        O₂ affinity and/or cooperativity of O₂ binding).

Thus, to minimize effects of facilitated diffusion on ΔO_(2T) fromcell-free hemoglobin-based oxygen carriers, a given ΔO_(2T) based on thevalue for red blood cells can be achieved using the above strategiesindependently or in combination. For the purpose of example, ΔO_(2T) canbe decreased to within a desired range by:

-   -   (1) altering a single parameter independently through:        -   increasing η;        -   increasing r_(HbO2);        -   decreasing [Hb]T or        -   adjusting ΔY through its O₂ affinity and/or cooperativity;    -   (2) altering any combination of the above properties such that,        quantitatively, ΔO_(2T) is within the desired range.        B. Evaluation of Cell-Free Hemoglobins

Evaluation of cell-free hemoglobins with regard to their facilitateddiffusion of oxygen and hence their potential to produce autoregulatoryvasoactivity in arterioles is based on the Krogh cylinder, an idealizedsegment of vessel (See FIG. 13). Through a detailed analysis of theshape and position of the oxygen equilibrium curve, the amount of O₂delivered to this sensitive region is analyzed as a function ofdiffusion, hemoglobin concentration, and P50.

1. Artificial Capillary System

The artificial capillary system is shown diagrammatically in FIG. 14.The capillary is polydimethlysiloxane (e.g., Silastic, Point MedicalCorporation, Crown Point, Ind.) with a wall thickness approximately thesame size as the diameter (57 μm). The glass capillary is a 2 μl pipette(e.g., Drummond Scientific, Broomall, Pa.). The glass and siliconejunction is sealed with a silicone sealant (e.g., RTV 60, GeneralElectric). The typical length of a capillary, 100 mm, produces residencetimes similar to in vivo times (i.e., 0.37 sec-1.5).

The infusion syringe pump (e.g., KD Scientific, Boston, Mass.) isconnected to the entry oxygen flow cell by a short length oflow-permeable Tygon tubing. The Clark-type oxygen electrodes (e.g.,Instech, Plymouth Meeting, Pa.) are used to monitor the system. Datacollection is accomplished by analysis of the effluent fluid with ablood-gas analyzer (ABL-5, Radiometer). The exit from the flow cell isconnected to another short length of Tygon tubing, which in turn istightened to the glass capillary of the silicone capillary unit by theuse of a micro-tube connector (e.g., Cole-Palmer, Niles, Ill.). Theartificial capillary is encased in a gas-tight exchange chamber made ofclear acrylic plastic. Bimetallic temperature probes (e.g., YSI 700,Yellow Springs, Ohio) are attached near the entry and exit points of thefluid flow to ensure proper temperature control and held constant at 37°C.

The collection cell is mated directly to the end of the artificialcapillary unit by use of a silicone sealant and a polypropylenemicrofitting (Cole-Palmer). The collection cell is solid acrylic with aT shaped channel (diameter of 0.75 mm) drilled through it. The firstchannel is shunted through a calibrated measuring tube that serves as aflow meter. Periodically the flow meter can be replaced with an oxygenelectrode to monitor system conditions. The second flow channel isdirected toward the back of the collection cell, where a gas-tightseptum seals the exit. A Hamilton gas-tight syringe (Hamilton Co., Reno,Nev.) pierces this septum and collects the sample as a syringe pumpslowly withdraws fluid at a rate lower than the flow rate in thecapillary. This entire apparatus is enclosed within an acrylic containerwhich maintains the temperature 37° C. through the use of a fin heater.

2. Artificial Capillary Experimental Protocol

The equilibrated samples are aspirated from the tonometer into aHamilton gas-tight syringe which is mounted onto the infusion pump.Constant flow is established throughout the system to achieve thedesired residence time. The test solutions are equilibrated with 20% O₂,balance N₂, to simulate air. The chamber outside of the capillary isfilled with 100% N₂. The inlet gas is routed through a 37° C. water bathand a flow meter to maintain constant flow rate, so that the volume ofgas in the chamber is exchanged every 10 seconds. Oxygen electrodesmonitor the extracapillary gas compartment.

The effluent from the capillary is collected in a second Hamiltongas-tight syringe and is injected into the blood gas analyzer (e.g.,ABL-5, Radiometer, West lake, OH). A minimum of three samples are takenat each residence time. Flow conditions are changed, and a new set ofsamples is tested. Three flow rates, 10, 20 and 40 μl/min, giveresidence times in the capillary of 1.56, 0.75 and 0.39 seconds,respectively.

3. Mathematical Analysis Of Artificial Capillary Data

For each segment (dx, FIG. 13) of distance along the capillary, thetotal O₂ present in the solution is:O ₂ _(r) =αPO ₂ +Y[Hb] _(T)where α is the solubility coefficient of O₂ in plasma (1.2074 μM/Torr)(Winslow et al, J. Biol. Chem., 252(7):2331-2337 [1977]), Y ishemoglobin saturation, and [Hb]_(T) is total hemoglobin concentration.The amount of O₂ transferred out of the capillary in the segment dx is$\begin{matrix}{{\Delta\quad O_{2}} = \frac{{K^{*}( {\Delta\quad P\quad O_{2}} )}( {\pi\quad r^{2}} )d\quad x}{R}} & (6)\end{matrix}$where K* is a lumped diffusion parameter, consisting of the diffusionconstants given in equation 1 and the length of the diffusion gradientfor 2. ΔPO₂ is the PO₂ gradient (essentially the interior PO₂ when N₂ isthe outside gas), r is the radius of the capillary, and R is the flowrate. Total O₂ is now decremented by ΔO₂. At this point, the Adairequation, using the known parameters for the hemoglobin in question, isused to empirically find the PO₂ and Y combination that provide the newO_(2T) according to equation (5). The process is repeated until the endof the capillary is reached, and the final PO₂ is matched with the valueactually measured in the experiment. A FORTRAN program was used toperform this analysis in finite elements of dx. Experiments wereconducted using these methods and devices, as described in theExperimental section below (See Example 16).C. Possible Modifications of Hemoglobin

No product currently under development can replace all the functions ofblood. Instead, these blood product solutions are distinguished fromother plasma expanders by their ability to increase the total oxygenthat can be delivered. Of these, there are two general types: those thatincrease dissolved oxygen (i.e., perfluorocarbons) and those that carryoxygen chemically bound to hemoglobin (hemoglobin-based O₂ carriers).There are significant differences between the two types and theytransport O₂ in fundamentally different ways.

Hemoglobin is a protein made up of 4 polypeptide subunits, 2 α and 2 βchains. One of each, tightly bound together, make up a half molecule (αβdimer) and two dimers are more loosely bound to form the fullyfunctional molecule (α₂β₂ tetramer). The interface between the αβ dimersslides apart as O₂ is reversibly bound, forming two structures, one eachcorresponding to the fully deoxygenated (T, tense) and one to the fullyoxygenated (R, relaxed) structure. These two conformers have vastlydifferent affinities for O₂ so that as O₂ molecules are sequentiallybound and the transition from deoxy to oxy occurs, the affinity for O₂increases. This change in affinity is called “cooperativity” and isrepresented by the Hill coefficient, n (FIG. 13).

The loose interface between αβ dimers is of critical importance forhemoglobin-based blood substitutes. The equilibrium constant for thisdissociation reaction is 10⁻⁶ M for HbO₂ which means that as hemoglobinconcentration falls, the relative proportion of dimeric moleculesincreases. These dimers are very quickly and efficiently filtered in theglomerulus of the kidney. Mechanisms to remove dimers which are presentwhen mild hemolysis occurs include haptoglobin binding which can removefree hemoglobin in concentrations up to 200 mg/dl. When this thresholdis exceeded renal clearance of hemoglobin is very high, and renaltoxicity may result.

Many chemical modifications of hemoglobin have been devised (See, Table3). The purposes of these modifications are to prevent tetramer-dimerdissociation, modulate oxygen affinity, and prolong vascular retention.They take advantage of several reactive sites on the surface ofhemoglobin, in its internal cavity and at the amino terminus. One of themost useful modifications for researchers (αα-hemoglobin, DCLHb™,HemAssist™) incorporates a single cross-link between a deoxyhemoglobinLysine 99 residues with the reagent DBBF (Walder et al., J. Mol. Biol.,141: 195-216 [1980]. This single modification at once binds αβ dimerstogether and reduces the O₂ affinity of cell-free molecules toapproximately that of intact human red blood cells. When crosslinking iscarried out with oxygenated hemoglobin, the dimensions of the internalcavity change enough so that the reaction occurs between β82 Lysines. Inthis case, the final crosslinked product has a much higher O₂ affinitythan that of the deoxy cross-linked product. This material can also beproduced easily, but has been less well studied because its O₂ affinityhas been traditionally thought to be too high to be physiologically orclinically useful. TABLE 3 Examples Of Hemoglobin Modifications UsefulIn Preparation Of Blood Substitutes Reagent/Modification Name ReferenceAmino acid modification: N-carboxymethylation DiDonato (1983) J. Biol.Chem., 258: 4 amino termini 11890-11895 monoisothiocyanate 2-, 3-,4-ICBS Currell (1994) Meth. Enzymol., 231: 281 4 amino termini pyridoxalphosphate PLP Benesch (1982) J. Biol. Chem., 257: Val-1(β) 1320-1324Cross-linked tetramers: mono-(3,5-dibromosalicyl) FMDA Bucci (1989) J.Biol. Chem., 264: 6191-6195 fumarate mono-(3,5-dibromosalicyl) Rayzynska(1996) Arch. Biochem. muconate Biophys., 325: 119-125bis(2,3-dibromo-salycyl) Bucci (1986) Biochim. Biophys. Acta fumarate874: 76-81 bis(3,5-dibromosalicyl) DBBF Walder (1979) Biochem., 18:4265-4270; fumarate ββ-Hb αα- and Chaterjee (1986) J. Biol. Chem.,Lys-82(β₁)-Lys-82(β₂) Hb, 261: 9929-9937 Lys-99(α₁)-Lys-99(α₂) DCLHb(HemAssist) bis-(3,5-dibromosalicyl) Bucci (1996) J. Lab. Clin. Med.,128: sebacate 146-153 2-nor-2-formylpyridoxal 5′- NFPLP Benesch (1981)Meth. Enzymol., phosphate 76: 147-158 Lys-82(β₁)-Val-1(β₂)bis(pyridoxal) diphosphate (bisPL)P2 Benesch (1988) BBRC 156: 9-14Lys-82(β₁)-Val-1(β₂) bis(pyridoxal) tetraphosphate (bisPL)P4 Benesch(1994) Meth. Enzymol., Lys-82(β₁)-Val-1(β₂) 231: 267 Diisothiocyanatobenzene DIBS Manning (1991) PNAS 88: 3329 sulfonate (α-DIBS-α)β2Val-1(α₁)-Val-1(α₂) diisothiocyanate Kavanaugh (1988) Biochem., 27: 804Trimesoyl tris(methyl Tm-Hb Kluger (1992) Biochem., 31: 7551-7559phosphate) β82-Hb Val-1(β₁)-Lys-82(β₁)-Lys- 82(β₂) Lys-82(β₁)-Lys-82(β₂)Recombinant dialpha fusion rHb 0.1 Looker (1992) Nature 356: 258-260wild type (Optro) βN108K (Presbyterian) Polymers: glycolaldehyde & Fantl(1987) Biochem., 26: 5755-5761 carboxymethylation glycolaldehyde & PLPMacDonald (1991) Eur Pat 9,104,011.3 glycolaldehyde & NFPLP MacDonald(1991) BACIB. 19: A424 glycolaldehyde & DBBF MacDonald (1994) Meth.Enzymol., 231: 287-308 glutaraldehyde (Hemopure), Lysines, N-termValines (Oxypure) glutaraldehyde & PLP PolyHeme DeVenuto (1982) Surg.Gyn. Obst., Lysines, N-term Valines SFH 155: 342-346 glutaraldehyde &NFPLP polyHbNFPLP Berbers (1991) J. Lab. Clin. Med., Lysines, N-termValines 117: 157-65 glutaraldehyde & DBBF Nelson (1992) BACIB 20:253-258 Oxidative ring-opened (Hemolink) Hsia (1989) US Pat 4,857,636raffinose Lysines, N-term Valines Surface Conjugates: cellulose Flemming(1973) Acta Biol. Med. Ger., 30: 177-182 dextran dialdehyde Tam (1976)Proc. Natl. Acad. Sci., 73: 2118-2121 dextran-alkylation Dx-Hb Chang(1977) Can. J. Biochem., Cys-93(β) 55: 398-403 dextran sulfate SF-DxBarberousse (1986) J. Chromatogr., 369: 244-247 dextran phosphate P-DxSacco (1990) Biochim. Biophys. Acta 1041: 279-284 dextran benzene Dx-BHCProuchayret (1992) BACIB 20: 319-322 hexacarboxylate hydroxyethyl starchCerny (1984) Appl. Biochem. Biotech., 10: 151-153 inulin Iwasaki (1983)BBRC 113: 513-518 polyvinylpyrrolidone Schmidt (1979) Klin. Wochenschr.57: 1169-1175 polyethylene glycol Ajisaka (1980) BBRC 97: 1076-1081methoxy-polyoxyethylene (PEG-Hb) Zalipsky (1991) Polymeric Drugs, pp91-100 (mPEG) 10-12 Lysines α-carboxymethyl, ω- (PHP) Iwashita (1995)Artificial Red Cells, pp carboxymethoxypolyoxyethylene 151-176(dicarboxyPEG) 8-10 Lysine & PLP

Another unique class of crosslinkers, trimesic acid derivatives, resultin 2- or 3-point reactions (Kluger et al., Biochem., 31:7551-7559[1992]). In early reports, the resulting modified hemoglobins producedwith these appeared to be stable and the reaction seemed to have a highdegree of specificity.

A variation on this 64,000 kD molecular weight hemoglobin is thegenetically produced “rHb1.1” (Looker et al., Nature 356:258-260 [1992])in which crosslinking is done genetically. In this case, 2 α chain genesare introduced into the E. coli genome such that when they aretranscribed, a single gene product results in which one α chain iscontiguous with the other (dialpha peptide). Thus, the product has amolecular weight of 64,000 kD and does not dissociate in to dimers. Itsphysiological properties are similar to αα-hemoglobin.

Other crosslinking agents are analogs of 2,3-DPG. NFPLP, a prototype ofsuch a crosslinker, binds in the 2,3-DPG “pocket” between β chains andhas the dual effects of preventing dimerization and reducing O₂affinity. This product has been extensively studied (Bleeker et al.,Biomater. Artific. Cells Immobil. Biotechnol., 20:747-750 [1992]) butunfortunately the crosslinker itself is difficult to synthesize, andscaleup has not been achieved practically.

Conjugated hemoglobins are those to which some modifying molecule hasbeen attached to the surface (See e.g., Nho et al., Biomat. Artif. CellsImmobil. Biotechnol., 20:511-524 [1992]). Modifying groups includepolyethylene glycol (PEG), polyoxyethylene (POE), or dextran. Theseproducts have increased molecular weights, depending on the number andsize of the modifying groups, but are relatively easy to produce.Increasing the molecular size may also increase the hydration shellaround the protein molecule, in the case of POE and PEG, and may therebyrestrict the reaction of hemoglobin with other molecules in thecell-free environment.

Finally, nonspecific reagents can react with any of the 44 the ε-aminolysine groups on the surface of hemoglobin or the 4 amino-terminalgroups. Such bifunctional reactants include glutaraldehyde ando-raffinose and have been used in at least three of the productspresently in clinical trials. While the modification reactions areclearly understood chemically, the extent of reaction can sometimes bedifficult to control, and a range of molecular weights of product mayresult (Marini et al., Biopolymers 29:871-882 [1990]).

The present invention provides methods to improve the currenthemoglobin-based red cell substitutes which have serious problems. Ingeneral, molecular size can be increased by polymerization of hemoglobinwith polyfunctional cross-linkers or by surface conjugation to polymerssuch as PEG, dextran, or other starches, carbohydrates, or proteins.Viscosity can be increased by conjugation to PEG or its analogues. Theviscosity of the solution can be increased by formulation with a highviscosity material such as pentastarch, dextrans, carbohydrates orproteins which are, themselves, viscous. Finally, oxygen affinity can beincreased by intramolecular crosslinking of hemoglobin in the Rconformational state. This can be achieved by placing the hemoglobin inan environment such as O₂, CO or other ligand which favors the Rconformation. Examples of specific changes to the production of modifiedhemoglobins to be used as cell-free oxygen carriers include thefollowing.

1. αα-Hemoglobin

This hemoglobin, initially designed as a model compound for study by theU.S. Army, has been produced by Baxter Healthcare and is being tested asa replacement for human blood in the immediate postoperative period andin selected trauma patients. Both the Army and Baxter have reported thatthis product produces significant elevations of blood pressure andvascular resistance, and preclinical animal studies have shown thatthese undesirable properties eliminate any advantage to be derived fromadministration of hemoglobin solution (See e.g., Hess et al., J. Appl.Physiol., 74:1769-1778 [1993]).

As presently formulated, αα-Hb has low viscosity (approximately 1 cP,shear rate of 160 s⁻¹, 37° C.), high [Hb] at approximately 10 g/dl, amolecular size that is the same as that of tetrameric hemoglobin, and ithas oxygen affinity similar to or lower than that of blood. The presentinvention provides αα-hemoglobin for which the viscosity has beenincreased by formulation in pentastarch or any high viscosity colloid.Indeed, the viscosity and molecular size can be increased by surfaceconjugation with PEG or any other suitable methods of surfacedecoration. In addition, the composition can be formulated with a lower[Hb]. In addition, its oxygen affinity can be increased by carrying outcrosslinking chemistry using DBBF with the starting hemoglobin materialin a (e.g., CO or O₂ liganded) high-affinity conformational state.

Thus, the present invention provides methods to improve this compositionby reducing its P50, for example by crosslinking the hemoglobin in aliganded (e.g., CO or O₂) state, and by increasing its molecular size bysurface decoration with PEG or other materials that increase itsmolecular radius and viscosity. Diffusion of O₂ in a solution ofαα-hemoglobin could be reduced by formulation in pentastarch.

2. rHb1.1

Recombinant hemoglobin (e.g., Optro™, Somatogen) may be produced usingvarious hosts (e.g., bacteria). Currently available recombinanthemoglobin consists, primarily, of fused α chains and the introductionof a mutation (Presbyterian) which reduces its oxygen affinity. Thepresent invention provides methods to improve this product by reducingits P50, for example, by eliminating the Presbyterian mutation or byintroducing other mutations that increase its oxygen affinity or reducecooperativity, and by increasing its molecular size by surfacedecoration with PEG or other materials that increase its molecularradius and viscosity. Diffusion of O₂ in a solution of Optro™ could bereduced by formulation in pentastarch.

As presently formulated, rHb1.1 has low viscosity, its molecular size isthat of tetrameric hemoglobin, and it has an oxygen affinity similar toor lower than that of blood. The present invention provides methods toincrease the viscosity of rHb1.1, by formulation in pentastarch or anyhigh viscosity colloid. In addition, its viscosity and molecular sizecan be increased by surface conjugation using surface conjugation withPEG or any other suitable methods of surface decoration. Furthermore, itcan be formulated with a low [Hb].

3. PHP (Pyridoxylated Hemoglobin Polyoxyethylene)

PHP (pyridoxylated hemoglobin polyoxyethylene; e.g., Apex Bioscience).This hemoglobin is from a human source, reacted with pyridoxal phosphate(PLP) to increase its P50, and then surface modified with a form ofpolyethylene glycol. The present invention provides methods to improvethis product by eliminating the PLP reaction, crosslinking the liganded(e.g., CO or O₂) state, and by more extensive surface decoration withPEG, either by increasing the number of PEG strands per molecule or byincreasing the length of individual PEG strands. Diffusion of O₂ in asolution of PHP could be reduced by formulation in pentastarch.

As presently formulated, PHP has an intermediate viscosity ofapproximately 2 cP (under conditions described herein), high [Hb] atapproximately 8 g/dl, its molecular size is slightly more than 2-foldlarger than a hemoglobin tetramer, and an oxygen affinity that isslightly greater than that of blood. The present invention providesmethods to increase the viscosity of this product by formulation inpentastarch or any high viscosity colloid. Its viscosity and molecularsize can be increased by more extensive surface decoration with PEG,either by increasing the number of PEG strands per molecule or byincreasing the length of individual PEG strands. It can also beformulated at lower [Hb], and/or its oxygen affinity increased, byeliminating PLP during hemoglobin modification reaction.

4. Hemolink™

Hemolink™ (Hemosol, Ltd.) is a human-derived hemoglobin product with avery high P50, and is polymerized with o-raffinose, a multifunctionalcrosslinking reagent. The present invention provides methods to improvethe product by crosslinking the liganded (e.g., CO or O₂) protein and byincreasing its molecular radius and viscosity. This could beaccomplished by surface decoration with any PEG derivative orconjugation to a polysaccharide or other polymer that would increase itsmolecular size. Diffusion of O₂ in a solution of Hemolink™ could bereduced by formulation in pentastarch.

As presently formulated, Hemolink has low viscosity (ca. 1.4 cP underconditions of our measurement), high [Hb] at approximately 10 g/dl, itsmolecular size is about 1.7-fold greater than tetrameric hemoglobin, andit has low oxygen affinity compared to blood. The present inventionprovides methods to increase the viscosity by formulation in pentastarchor any high viscosity colloid. Its viscosity and molecular size can beincreased by surface conjugation using surface conjugation with PEG orother methods of surface decoration. In addition, it can be formulatedat lower [Hb]. Its oxygen affinity can be increased by carrying out itspolymerization chemistry using o-raffinose with the starting hemoglobinmaterial in a (e.g., CO or O₂ liganded) high-affinity conformationalstate.

5. HemoPure™

HemoPure™ (Bio-Pure) is a bovine-derived hemoglobin product with amoderately high P50, and is polymerized with glutaraldehyde, abifunctional crosslinking reagent. The present invention providesmethods to improve the product by crosslinking the liganded (e.g., CO orO₂) protein and by increasing its molecular radius and viscosity. Thiscould be accomplished by surface decoration with any PEG derivative orconjugation to a polysaccharide or other polymer that would increase itsmolecular size. Diffusion of O₂ in a solution of HemoPure™ could bereduced by formulation in pentastarch.

6. Polyheme™

Polyheme™ (Northfield Laboratories) is a human-derived hemoglobinproduct with a moderately high P50 due to reaction with PLP, and ispolymerized with glutaraldehyde, a bifunctional crosslinking reagent.The present invention provides methods to improve the product bycrosslinking the liganded (e.g., CO or O₂) protein, eliminating the PLPand by increasing its molecular radius and viscosity. This could beaccomplished by surface decoration with any PEG derivative orconjugation to a polysaccharide or other polymer that would increase itsmolecular size. Diffusion of O₂ in a solution of Polyheme™ could bereduced by formulation in pentastarch.

As presently formulated, Polyheme has high [Hb] at approximately 10g/dl, its molecular size is larger than that of tetrameric hemoglobin bybeing polymerized, and its oxygen affinity is lowered by reaction withPLP. The present invention provides methods to increase the viscosity byformulation in pentastarch or any high viscosity colloid. Its viscosityand molecular size can be increased by surface conjugation using surfaceconjugation with PEG or other methods of surface decoration. It can alsobe formulated at lower [Hb]. Its oxygen affinity can be increased bycarrying out its polymerization chemistry using glutaraldehyde in theabsence of PLP and with the starting hemoglobin material in a (e.g., COor O₂ liganded) high-affinity conformational state.

7. PEG-Hb

Commercially available PEG-Hb compositions (e.g., Enzon) may be improvedusing the present invention by decreasing its concentration and byformulation in pentastarch. Enzon's hemoglobin consists of a bovinehemoglobin modified by conjugation to linear 5000 MW polyethylene glycol(PEG) chains. Polyalkylene oxide (PAO) is a generic term for a group ofmolecules that includes PEG. Attachment of PAO to hemoglobin is achievedby formation of a covalent bond between the PAO and the ε-amino groupsof lysine residues. Enzon's hemoglobin is conjugated to 10-12 PAO chainsper hemoglobin tetramer. When measured at a shear rate of 160 s⁻¹, 37°C., a 5 g/dl solution of Enzon's PEG-Hb exhibits a viscosity of 3.39 cP.

As presently formulated, PEG-Hb has a viscosity of approximately 3.5 cp(under conditions of described herein) at a [Hb] of 5.5 g/dl, itsmolecular size is 4-fold greater than that of tetrameric hemoglobin, andit has high oxygen affinity relative to blood. The present inventionprovides methods to increase the viscosity of this product at lower [Hb]by formulation in pentastarch or any high viscosity colloid.Furthermore, its viscosity and molecular size can be increased by moreextensive surface decoration with PEG, either by increasing the numberof PEG strands per molecule or by increasing the length of individualPEG strands. It can also be formulated at lower [Hb].

Additional methods to increase the viscosity (η, cP) unit per unitconcentration ([Hb], g/dl) of a hemoglobin solution include, but are notlimited to the following:

1. Increase the Number of Sites Conjugated to 5000 MW PAO Per HemoglobinTetramer

Human hemoglobin contains 44 lysine residues (11 on each chain). Incombination with the 4 N-terminal amino groups, this gives 48theoretically possible sites for covalent attachment of PAO using thechemistry described for modification of amino groups. Additionalchemistry has been described (See e.g., Acharya's U.S. Pat. No.5,585,484; herein incorporated by reference) that allows covalentattachment of PAO to the sulfhydryl group of a cysteine residue. Thereare 6 cysteine residues per Hb tetramer (i.e., one on each α chain, andtwo on each β chain) increasing the number of theoretically possibleattachments to 54. Further PAO modifications are contemplated, includingthe use of suitable conjugation chemistry lead to attachment to serine,threonine, tyrosine, asparagine, glutamine, arginine, and histidineresidues. It is also contemplated that chemistry that allows conjugationto carboxylic acid groups may allow PAO conjugation to aspartic acid andglutamic acid residues as well as the C-terminal carboxy groups ofhemoglobin.

If the number of conjugation sites per tetramer is sufficiently large,it is contemplated that PAO molecules of lower molecular mass (i.e.,MW<5000) will still achieve an increased viscosity per unitconcentration over Enzon's product without the modifications describedherein.

2. Maintain the Number of Covalent PAO Attachments and Increase the Sizeof Each PAO Moiety

An increased viscosity per unit hemoglobin concentration is contemplatedin situations in which the PAO groups attached to the 10-12 sites pertetramer are of greater molecular mass (i.e., MW>5000). This can beachieved by using PAO starting material consisting of moleculescontaining longer and/or branched PAO chains.

If the molecular size of the PAO units is sufficiently large, it may bepossible to modify a fewer number of sites on the tetramer (i.e., <10)and still achieve an increased viscosity per unit concentration overEnzon's product.

IV. The Oxygen-Carrying Component of the Blood Products of the PresentInvention

In preferred embodiments of the present invention, the oxygen-carryingcomponent is native or modified hemoglobin (e.g., a HBOC). Modifiedhemoglobin is altered by chemical reaction (e.g., cross-linking orpolymerization) or through the addition of adducts (e.g.,polyethyleneglycol, polyoxyethylene). Furthermore, the oxygen-carryingcomponent of the present invention may be recombinantly-producedhemoglobin or a hemoglobin product encapsulated in a liposome. Thepresent invention also contemplates the use of other means for oxygendelivery that do not entail hemoglobin or modified hemoglobin.

Though the present invention contemplates the use of any oxygen-carryingcomponent, preferred oxygen-carrying components entail solutions ofhuman or animal (e.g., bovine) hemoglobin, intramolecularly crosslinkedto prevent dissociation into dimeric form. Optionally, the preferredoxygen-carrying components of the present invention may be oligomerizedto oligomers of molecular weight up to about 750,000 daltons, preferablyup to about 500,000 daltons. Hemoglobin preparations prepared by geneticengineering and recombinant processes are also among the preferredoxygen-carrying components.

The preferred oxygen-carrying components of the present invention shouldbe stroma free and endotoxin free. Representative examples of preferredoxygen-carrying components are disclosed in a number of issued UnitedStates Patents, including U.S. Pat. No. 4,857,636 to Hsia; U.S. Pat. No.4,600,531 to Walder, U.S. Pat. No. 4,061,736 to Morris et al.; U.S. Pat.No. 3,925,344 to Mazur; U.S. Pat. No. 4,529,719 to Tye; U.S. Pat. No.4,473,496 to Scannon; U.S. Pat. No. 4,584,130 to Bocci et al.; U.S. Pat.No. 5,250,665 to Kluger et al.; U.S. Pat. No. 5,028,588 to Hoffman etal.; and U.S. Pat. No. 4,826,811 and U.S. Pat. No. 5,194,590 to Sehgalet al.; the contents of each are hereby incorporated by reference. In amore preferred embodiment, the oxygen-carrying components comprisehuman, recombinant, or animal hemoglobin, either cross-linked or not,modified by reaction with polyethyleneglycol (PEG) or polyoxyethylene(POE).

The capacity of a solution to deliver oxygen to tissues can bedetermined in a number of ways routinely used by researchers, includingdirect measurement of oxygen tension in tissues, increased mixed venousoxygen tension, and reduced oxygen extraction ratio.

V. The Non-Oxygen-Carrying Component of the Blood Products of thePresent Invention

As noted above, the present invention contemplates a mixture comprisingan oxygen-carrying component and a non-oxygen-carrying component. Thenon-oxygen-carrying component of the present invention is any substanceused for temporary replacement of RBCs which has oncotic pressure (e.g.,dextran-70, dextran-90, hespan, pentastarch, hetastarch, albumin, or anyother colloidal intravenous solution).

Non-oxygen-carrying plasma expander products for the treatment ofhypovolemia and other conditions are commercially available;representative products include, but are not limited to, Pentaspan®(DuPont Merck, Fresenius), Hespan® (6% hetastarch in 0.9% sodiumchloride for injection; DuPont Merck), and Macrodex® (6% Dextran 70 in5% dextrose in water for injection, or 6% Dextran 70 in 0.9% sodiumchloride for injection; Pharmacia). Non-oxygen-carrying fluids availablefor clinical use (e.g., hemodilution or resuscitation) can be broadlyclassified as crystalloid solutions (i.e., salt solutions) and colloidsolutions. In preferred embodiments of the present invention, colloidsolutions comprise the non-oxygen-carrying component of the mixture.

In one embodiment of the present invention, the problems of the priorart products are alleviated by the formulation and use of a composition(an aqueous solution) that contains both an oxygen-carrying component(e.g., a HBOC) and a non-oxygen-carrying component comprising an inert,non-proteinaceous colloid. Such compositions result in two effects,either alone or in combination. First, the oxygen carrying capacity ofthe composition is decreased, while colloid osmotic (oncotic) pressureand plasma retention are maintained. The resulting colloid-dilutedoxygen-carrying component has fewer oxygen-delivering colloidalparticles per unit volume than the oxygen-carrying component alone, andhence there is less oxygen presented to the arterial walls. That is, theoxygen delivery more closely approximates that of whole blood, so thatthe combination according to the invention is able to deliver anddistribute its oxygen loading in a manner more closely resembling thatachieved by RBCs.

Second, by proper choice of type and amount of non-proteinaceous colloid(discussed below), the viscosity of an oxygen-carrying component-colloidcomposition can be increased, preferably close to that of whole blood.This also appears to reduce or counteract arterial wall reaction. Thoughan understanding of the mechanism of this effect is not required inorder to practice the present invention, it is believed to be due to i)reduced oxygen delivery as a result of decreased hemoglobin and ii)increased shear stress at the vessel wall (which results in theincreased release of endogenous vasodilators such as prostacyclin).

Suitable examples of non-proteinaceous colloids for use in thecompositions of the present invention include dextran andpharmaceutically-acceptable derivatives thereof, starch andpharmaceutically acceptable derivatives thereof, andpolyvinylpyrrolidone (PVP). Particularly preferred among suitablenon-proteinaceous colloids is pentastarch. Indeed, suitablenon-proteinaceous colloids include substantially all non-proteinaceouscolloidal substances which have previously been successfully used ashemodiluents. Acceptable candidates should be water soluble, exhibitoncotic pressure, and be biologically inert and otherwisepharmaceutically acceptable. The cost of these materials (e.g., oncoticnon-proteinaceous colloids like dextran and hetastarch), on a weight forweight basis, is much lower than that of hemoglobin and HBOCs.

III. Clinical and Other Applications of the Present Invention

The present invention finds use in many settings, ranging from theemergency room to the operating table, as well as military conflicts,and veterinary applications. This versatility is due to the optimizedformulations of the present invention, which may be stored as desired,and avoid the necessity for cross-matching or other laboratory tests todetermine compatibility with the patient to be treated. Extensiveresearch on chemical and genetic modifications of hemoglobin, inconjunction with the present invention now permit the design ofmolecules with nearly any desired combination of physical andphysiological properties in a heretofore unexpected and highly efficientmanner.

A. Clinical Applications

Various clinical applications are matched with properties of theproposed red cell substitutes in Table 4, below. In this Table, T_(1/2)refers to the half-life. TABLE 4 Potential Clinical Applications For RedCell Substitutes And Optimal Properties Appilcation COP P50 ViscosityT_(1/2) Hemodilution ↑ ↓ ↑ ↑ Trauma ↑ ↓ ↑ ↑ Septic Shock ↑ ↓ ↑ ↑Ischemia (e.g., stroke) ↑ ↓ ? ↓ Cancer — ↓ ↓ ↑ Chronic Anemia — ↓ ↑ ↑Sickle Cell Anemia ↑ ↑ ↓ ↑ Cardioplegia ↑ ↑ ↓ — Hypoxia — ↓ ↑ ↑ OrganPerfusion — ↑ ? — Cell Culture — — — — Hematopoiesis ↓ ↑ ↓ ↓

It is contemplated that high oncotic activity (COP) will find use in theshort term, immediate, resuscitation from hypovolemic shock. The utilityof hypertonic saline/dextran (HSD) has been shown in animal studies(Kramer et al., Surgery, 100(2):239-47 [1986]). Oncotic activity (COP)expands the vascular volume very quickly and it is contemplated thatperhaps this, combined with the rapid restoration of O₂ capacity, mightlead to significantly better salvage of patients and tissues after acuteblood loss. However, there numerous settings in which the compositionsand methods of the present invention find use including the following:

Hemodilution. In this clinical application, the patient comes to surgeryand some volume of blood is removed, to be replaced with the substitute.The goal is preventative, not to correct some imbalance. A solution thatperforms very close to blood is needed. A slightly increased COP isdesired because it increases blood volume and cardiac output, inanticipation of surgical blood loss. Since the replacement fluid ishemoglobin-based, a reduced P50 is preferred, in order to overcomefacilitated diffusion. Viscosity should be increased for the samereason, and the T_(1/2) should be prolonged to eliminate or reduce theneed for postoperative transfusion with allogeneic blood units, shouldthe ones collected prior to surgery (autologous) not be sufficient. Thesolution for hemodilution would have the same properties as one used incardiopulmonary bypass.

Trauma. In trauma, the patient has lost whole blood. In response to thisblood loss, fluid shifts from the interstitial and intracellular spacesto attempt to replace lost volume. In the process, hematocrit andviscosity fall and vasoconstriction occurs to shunt blood from organsthat have low priority. These include the skin and gut, for example,while blood flow to the kidneys, heart and brain are preserved for aslong as possible. The goal of a therapeutic blood replacement here wouldbe to first replace lost volume as fast as possible. Hence, increasedCOP are desired. Since the replacement fluid is hemoglobin-based, areduced P50 is preferred, in order to overcome facilitated diffusion.The viscosity should be increased for the same reason.

Septic Shock. In overwhelming sepsis, some patients may becomehypertensive in spite of massive fluid therapy and treatment withvasoconstrictor drugs. The mechanism of lowered blood pressure in thisinstance is overproduction of nitric oxide (NO). Therefore hemoglobin isclose to an ideal agent to treat these patients with because of theavidity with which hemoglobin binds NO. In general, NO binding affinityparallels O₂ binding affinity, so an agent for use in this applicationshould have very high O₂ affinity (low P50). Since the patients areoften fluid overloaded, increased COP would be desired, but notessential, and increased viscosity would reduce autoregulatoryvasoconstriction. The T_(1/2) should be moderately long, but it is notnecessary to be markedly prolonged, since continuous infusions can beused in these patients.

Ischemia (e.g., stroke). Ischemia refers to the condition where tissueis “starved” for oxygen. This usually results from limitation of bloodflow as in, for example, a heart attack or cerebrovascular accident. Thetissue, starved of O₂ dies in small patches, called “infarcts.” The goalof blood replacement therapy here would be to increase blood flow and topromote O₂ delivery into capillary beds. Hence, a solution of lowerviscosity may be preferred, in order to better perfuse capillary beds.This can be done only if the blood volume is maintained or expanded, andtherefore an increased COP would be desirable. In most situations ofheart attack and stroke, the tissue damage is acute, so therapy is onlynecessary for a few hours. Thus, the T_(1/2) is less important than inother applications.

Cancer. To increase the radiosensitivity (or sensitivity tochemotherapy), the goal is to deliver as much O₂ to the hypoxic core ofthe tumor as possible. The microcirculation of tumors is unlike that ofother tissues, because it lacks endothelial lining of capillaries, andnormal vasoactivity does not occur. Thus, it should be possible toprovide solutions of low viscosity. The P50 should be very low so thatlittle, if any, O₂ is unloaded in tissues before it reaches the hypoxiccore of the tumor. In other words, we would like O₂ to be unloaded atvery low PO₂, if possible Plasma T_(1/2) can be as long as possible, sothat repeated doses of irradiation or chemotherapy can be administered.

Chronic anemia. These patients are unable to regenerate lost red cellsor they are not able to keep production up with normal (or accelerated)destruction. In this situation, it is desired that the transfusionsubstitute to behave as much as possible like native red cells. Thus,facilitated diffusion should be overcome by increasing oxygen affinityand viscosity. In this application, more than any other, the T_(1/2) isvery important because patients will be unable to replace lost ormetabolized hemoglobin on their own.

Sickle cell anemia. This is a unique clinical condition in that red cellturnover is very high, and the sickling process in the affected person'sred cells is a function of PO₂. That is, the lower the PO₂, the greaterthe sickling rate. Sickling is also a function of red cell density andviscosity, which, in turn, is strongly dependent on hematocrit. Theideal solution in a sickle cell crisis would be one that delivers O₂ tosickled red cells. Thus, it may be preferable to use a high, rather thanlow, P50 so there is a net transfer of O₂ in favor of the sickled redcells. In order to do this, it would be necessary to decease diffusionin any way possible, to reduce vasoactivity which could offset anypotential benefit of oxygenating the red cells. At the same time, it ispreferred that the solution to have good flow properties. Thus, abalance between P50 and viscosity would have to be struck such that redcells are oxygenated while vasoconstriction is blocked or, at least, notinduced.

Cardioplegia. In certain cardiac surgical procedures, the heart isstopped by appropriate electrolyte solutions and reducing thetemperature of the patient. Reduction of the temperature will reduce P50drastically, possibly to the point where O₂ may not be unloaded underany ordinary physiological conditions. Thus, the P50 of a solution forthis purpose might be higher than for other applications. The viscosityis also temperature-dependent and appropriate adjustments would be madesuch that the in vivo viscosity is close to that of blood under thespecific conditions of the patient.

Hypoxia. In altitude dwellers and world-class athletes and soldiersunder extreme conditions, extraction of O₂ from air in the lung maybecome limiting to overall O₂ transport. This aspect of O₂ transportwould probably be more important than the ability of the solution tounload O₂ in tissues. In this case, lower P50 would be advantageous, andcooperativity should be maximal. Vasoactivity would not be desired, soviscosity would be elevated. The COP of such solutions would not need tobe elevated, and the plasma T_(1/2) should be as long as possible.

Organ Perfusion. Here, the main goal is to increase O₂ content of theperfusate. The parameters of O₂ loading and unloading are less importantthan in other conditions, since the fluid is not flowing. Therefore,nearly complete extraction is possible. P50 can be relatively normal oreven elevated, since the solutions can be oxygenated with externaloxygenators.

Cell Culture. This requirement is almost identical to that of organperfusion, except that the rate of O₂ consumption may be higher,depending on the cells and their concentration.

Hematopoiesis. Here, the hemoglobin is serving as a source of heme andiron, to be resynthesized into new hemoglobin. Thus, the hemoglobinshould be taken up into the monocyte-macrophage system and broken downin such a way as to make its components available for red cellmetabolism and maturation. The properties of COP, P50 and viscosity canbe the same as the hemodilution solution. The T_(1/2) can be relativelyshort, as long as metabolism is efficient.

Many workers in the field of oxygen transport have assumed that oxygenaffinity of modified hemoglobin should be low, or at least notsignificantly different from that of red cells, in order to maximizetissue oxygenation. During the development of the present invention, itwas found that this concept is invalid. In severe hypoxia, pulmonary O₂diffusion may become limiting to O₂ uptake in the alveolus, asdemonstrated in mountaineers at extreme altitude (Winslow et al.,[1984]). In this instance, increased, rather than decreased O₂ affinityis beneficial because it increases arterial O₂ saturation. Based on thehigh altitude data, this point is reached at approximately 6,000 metersaltitude, or at a PaO₂ of about 40 Torr. By extrapolation, one mightconclude that sea level patients with severe restrictions in diffusivepulmonary O₂ uptake might also benefit from increased hemoglobin O₂affinity. If the pulmonary capillary PO₂ reaches a maximal value of 40Torr (or less), then shifting the oxygen equilibrium curve to the leftwill increase saturation, in effect providing the same increase in O₂content as a transfusion, without adding the burden of increased redcell mass and, hence, viscosity.

In general, plasma retention times should be as long as possible.However, it is also contemplated that perhaps for O₂ delivery tospecific tissues (e.g., tumors, myocardium, ulcers, sickle cell disease)this property might not be so important. Furthermore, if the reason togive a hemoglobin solution is to stimulate erythropoiesis, it iscontemplated that a short retention time is desired.

The present invention provides data that show if the properties ofviscosity, oncotic pressure, oxygen affinity and hemoglobinconcentration are optimized as described, the hemoglobin can beformulated with additional components to serve additional functions ofblood. For example, coagulation factors (e.g., Factors VIII, IX, and/orII), immunoglobulins, antioxidants, iron chelators, peroxidases,catalase, superoxide dismutase, carbonic anhydrase, and other enzymesmay be mixed with the hemoglobin solution in order to provide benefit topatients in need of such compositions. Similarly, drugs such ascytotoxins, antibiotics or other agents may be mixed with the solutionor chemically conjugated to other components, such as hemoglobin orother polymers.

In addition, the final product can be formulated at any desiredelectrolyte and salt composition. It can be stored in the liquid state,frozen or lyophilized as the final product or the hemoglobin componentitself can reconstituted with any solution subsequently. Suchreconstitution medium could be, but need not be limited to, saline,Ringer's lactate, albumin solution, or PlasmaLyte, for example. Thefinal product can be stored in any biocompatible container such as glassor plastic.

B. Veterinary Applications

The present invention is not limited to use in humans. In addition tothe clinical applications briefly described above, the present inventionfinds utility in the veterinary arena. The compositions of the presentinvention may be used with domestic animals such as livestock andcompanion animals (e.g., dogs, cats, birds, reptiles), as well asanimals in aquaria, zoos, oceanaria, and other facilities that houseanimals. For example, as with humans, the compositions of the presentinvention may be used for emergency treatment of domestic and wildanimals traumatized by blood loss due to injury, hemolytic anemias, etc.For example, it is contemplated that embodiments of the presentinvention in such as equine infectious anemia, feline infectious anemia,hemolytic anemias due to chemicals and other physical agents, bacterialinfection, Factor IV fragmentation, hypersplenation and splenomegaly,hemorrhagic syndrome in poultry, hypoplastic anemia, aplastic anemia,idiopathic immune hemolytic conditions, iron deficiency, isoimmunehemolytic anemia, microangiopathic hemolytic, parasitism, etc.). Inparticular, the present invention finds use in areas where blood donorsfor animals of rare and/or exotic species are difficult to find.

VI. Blood Product Compositions

The relative proportions of the oxygen-carrying component and thenon-oxygen-carrying component (e.g., a colloid plasma expander) includedin the compositions of the present invention can vary over wide ranges.Of course, the relative proportions are, to some extent, dependent uponthe nature of the particular components, such as the molecular weight ofthe colloid used as a non-oxygen-carrying plasma expander. However, thepresent invention is not limited to the use of colloids as thenon-oxygen-carrying component.

In preferred embodiments of the present invention, the hemodilutioneffect of the non-oxygen-carrying component (e.g., a non-proteinaceouscolloid) predominates, i.e., the overall oxygen-carrying capacity of theoxygen-carrying component is reduced by dilution so that the adverseeffects of excessive oxygen release at the arterial walls arealleviated. In such embodiments, substantial economic benefits arederived from a composition that preferably contains at least 20% byweight of each of the components, and more preferably at least 25% byweight of each component. Most preferable compositions comprise fromapproximately 30 to approximately 70 parts of the oxygen-carryingcomponent (e.g., HBOC), correspondingly, from approximately 70 toapproximately 30 parts of the non-oxygen carrying component (e.g., inertcolloid) (per 100 parts by weight of the combination of the two).

In preferred embodiments, the viscosity of the blood substitutecompositions of the present invention is preferably close to that ofnormal blood. Thus, when it is desirable to utilize a composition whoseprimary purpose is to increase viscosity, high molecular weight colloidsin amounts of from approximately 1 to approximately 20 parts by weightper 100 parts by weight of oxygen-carrying component are preferred.

In other preferred embodiments of the present invention, increasedviscosity (i.e., to a value approaching that of whole blood) of thecomposition is the predominant effect. In these compositions, theviscosity of the composition is high enough so that shear stresses atthe arterial walls are sufficient to release endogenous vasodilators tocounteract the effects of the plentiful oxygen availability at thearterial walls. In such embodiments, the non-oxygen carrying component(e.g., non-proteinaceous colloid) should have a substantially highermolecular weight than the oxygen carrying-component, but should be usedin smaller amounts to avoid excessive viscosities. Polyvinylpyrrolidone(PVP) of molecular weight 300,000-750,000 used in amounts from about 1to about 20 parts by weight per 100 parts by weight of oxygen-carryingcomponent is particularly suitable in these embodiments. Similarly, highmolecular weight starches (e.g., approximately 200,000-750,000 molecularweight) are also preferred in these embodiments. The amounts are chosenso as to result in an oxygen-carrying component—colloid solution havinga viscosity, relative to whole blood (assigned a value of 1), of fromabout 0.5 to about 1.2.

In certain embodiments of the present invention, advantage is taken ofboth of the above-mentioned effects. That is, an amount and type of thenon-oxygen-carrying component (e.g., non-proteinaceous inert colloidplasma expander) is chosen which both reduces the amount of oxygencarried by a unit volume of the solution, and increases its viscosity toa level approximating that of normal whole blood. For this purpose, PVPand starches of molecular weights higher than that of theoxygen-carrying component are used, and in amounts sufficient toincrease the viscosity, to reduce the amount of oxygen carried, and toreduce the cost of the solution. Specifically, PVP and starchespossessing molecular weights from about 200,000 to about 600,000 used inamounts from about 5 to about 50 parts by weight of inert colloid per100 parts by weight of the oxygen-carrying component are contemplatedfor use with the present invention.

In some embodiments, the present invention contemplates that theconcentration of the combined oxygen-carrying component andnon-oxygen-carrying component (e.g., inert colloid plasma expander) inthe aqueous solution compositions will generally be in the same range asthat usually employed when one of the ingredients is used alone for thesame purpose (i.e., from about 5 to about 15 grams of the combinationper decaliter of solution).

The compositions of the present invention provide the followingimprovements over current blood substitutes: i) decreased concentrationof hemoglobin to which the patient is exposed, thereby reducing thetoxicity and cost of the blood product; ii) oncotic pressure, which moreeffectively expands the vascular volume than the currently used bloodsubstitutes; iii) optimal viscosity which maintains capillary bloodflow; iv) optimal oxygen affinity which reduces oversupply of oxygen toarteriolar walls; and v) optimal oxygen carrying capacity. All of theseimprovements increase the effectiveness of the blood products as acell-free oxygen carrier.

Several prior art references discuss the possibility of mixinghemoglobin solutions with non-oxygen carrying plasma expanders. Forexample, U.S. Pat. No. 4,061,736 to Morris et al. and U.S. Pat. No.4,001,401 to Bonson et al. describe pharmaceutical compositionscomprising an analog of hemoglobin and a pharmaceutically acceptablecarrier; the carrier may comprise, for example, polymeric plasmasubstitutes (e.g., polyethylene oxide). Similarly, U.S. Pat. No.5,349,054 to Bonaventura et al. describes a pharmaceutical compositioncomprising a hemoglobin analog which can be mixed with a polymericplasma substitute (e.g., polyvinylpyrrolidone). However, the prior artdoes not describe the specific compositions nor the techniques of thepresent invention for improving the effectiveness of a blood substituteand reducing the toxicity of those solutions.

DESCRIPTION OF SOME PREFERRED EMBODIMENTS

Generally speaking, compositions comprising i) an oxygen-carryingcomponent (e.g., a HBOC) with high oncotic pressure, oxygen affinity andviscosity and ii) a non-oxygen-carrying component with similar oncoticpressure and viscosity provide an optimal blood product. In the mostpreferred embodiments of the present invention, the oxygen-carryingcomponent of the mixture comprises a polyethylene glycol-modifiedhemoglobin and the non-oxygen-carrying component comprises pentastarch.

As described in more detail in the Experimental section, there arecurrently two commercially available hemoglobin products modified withpolyethylene glycol. The first product, Pyridoxal HemoglobinPolyoxyethylene (PHP), is a human-derived product from Apex Bioscience.The second product, PEG-Hb, is a bovine-based product obtained fromEnzon, Inc. Though most of the experimental work was performed usingPEG-Hb, the two PEG-modified hemoglobin products gave qualitatively thesame results. It is to be understood that the preferred oxygen-carryingcomponents of the present invention are not limited to PEG-Hb and PHP;indeed, any hemoglobin products associated with polyethylene glycol arecontemplated for use with the most preferred mixtures of the presentinvention.

Pentastarch, the most preferred non-oxygen-carrying component of thepresent invention, is commercially available from DuPont Merck(Pentaspan®) as well as from other sources. It comprises hydroxethylstarch and has a molecular weight of approximately 250,000 Daltons.Because of its lower molecular weight and lower degree of hydroxyethylsubstitution compared to other starches (e.g., hetastarch), it exhibitshigher oncotic pressure and faster enzymatic degradation in thecirculation. As described in detail in the Experimental section,dilution of PEG-Hb with a different non-oxygen-carrying component likehetastarch reduces the resulting blood product's viscosity and oncoticpressure, and reduces the oxygen capacity of the resulting mixture. Incontrast, the mixtures resulting from combination of PEG-modifiedhemoglobin with pentastarch have viscosity and oncotic pressure valuesvery close to that of PEG-Hb alone, and have been shown to lead toenhanced animal survival and physiological parameters compared to othermixtures (see Experimental section).

Preferred mixtures of polyethylene glycol-modified hemoglobin andpentastarch contain at least 20% by weight of each of the components,and more preferably at least 25% by weight of each component. Mostpreferable compositions comprise from approximately 30 to approximately70 parts of the oxygen-carrying component PEG-modified hemoglobin, and,correspondingly, from approximately 70 to approximately 30 parts of thenon-oxygen carrying component pentastarch (per 100 parts by weight ofthe combination of the two).

The experimental results presented below indicate that a mixture ofPEG-Hb and pentastarch performed similarly to a solution of PEG-Hbalone. This was true even though the hemoglobin concentration to whichthe animals were exposed and the amount of hemoglobin product used wereless by half with the mixture, offering the advantage of reducing theconcentration of hemoglobin given to patients, thereby reducing bothcost and potential adverse effects.

As previously indicated, the compositions and methods of the presentinvention can be used in any situation in which banked blood iscurrently administered to patients. For example, the compositions can beadministered to patients who have lost blood during surgery or due totraumatic injury. The compositions of the present invention areadvantageous in that they save the patient exposure to possibleinfectious agents, such as human immunodeficiency virus and hepatitisvirus.

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); mM (millimolar); μM(micromolar); g (grams); mg (milligrams); μg (micrograms); kg(kilograms); L (liters); mL (milliliters); dL (deciliters); μL(microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm(nanometers); min. (minutes); s and sec. (seconds); b.w. (body weight);i.p. (intraperitoneal or intraperitoneally); Da (Daltons); dP/dt (changein pressure over time); IU (international units); Hg (mercury); Hz(hertz); MHz (mega hertz); COP (colloid osmotic pressure); CRBCv(Capillary red blood cell velocity); FCD (functional capillary density);FDA (United States Food and Drug Administration); Hb (hemoglobin); MAP(mean arterial pressure); Pd (palladium); PEG (polyethylene glycol);PEGHb (bovine hemoglobin modified by conjugation with polyethyleneglycol); sat. (saturation); sem and s.e.m. (standard error of the mean);TM (trimesic acid); Abbott (Abbott Laboratories, Chicago, Ill.); Beckman(Beckman Instruments, Fullerton, Calif.); Bectron (NJ); Dupont (DupontPharmaceuticals, Wilmington, Del.); EG&G Electro Optics (Salem, Mass.);Enzon, Inc., (Piscataway, N.J.); Fresenius (Walnut Creek, Calif.);Hemocue, Inc. (Mission Viejo, Calif.); Hemosol Inc. (Etobicoke, ON,Canada); IPM (IPM, Inc., San Diego, Calif.); Lexington Instruments(Waltham, Mass.); Pharmacia (Pharmacia, Inc., Piscataway, N.J.);Porphyrin Products, Inc. (Logan, Utah); Sharp (Japan); Sony (Japan); TCSMedical Products (Hintingdon Valley, Pa.); Tektronix (Tektronix Inc.,Beaverton, Oreg.); Wescor (Logan, Utah).

The following general methods were used in the examples that followunless otherwise indicated.

Animal Model And Preparation

Experiments (except those described in Example 16) were carried out with10 Syrian golden hamsters of 40-50 g body weight. A “hamster windowpreparation” was then generated in each animal using a describedsurgical technique. (See, e.g., H. D. Papenfuss et al., “A transparentaccess chamber for the rat dorsal skin fold,” Microvasc. Res. 18:311-318[1979]; H. Kerger et al., “Systemic and subcutaneous microvascularoxygen tension in conscious Syrian golden hamsters,” Am. J. Physiol.,267 (Heart. Circ. Physiol. 37):H802-810 [1995]). Briefly, each animal'sdorsal skinfold, consisting of 2 layers of skin and muscle tissue, wasfitted with two titanium frames with a 15 mm circular opening andsurgically installed under pentobarbital anesthesia (50 mg/kg b.w.,i.p., Membutal®, Abbott). Layers of skin muscle were carefully separatedfrom the subcutaneous tissue and removed until a thin monolayer ofmuscle and one layer of intact skin remained.

Thereafter, a cover glass held by one frame was placed on the exposedtissue, allowing intravital observation of the microvasculature. Thesecond frame was open, exposing the intact skin. PE10 catheters wereimplanted in the jugular vein and the carotid artery. The catheters werepassed subcutaneously from the ventral to the dorsal side of the neck,and exteriorized through the skin at the base of the chamber. Thepatency of the catheters was ensured by daily flushing of the implantedtip with 0.005-0.01 mL of heparinized-saline (40 IU/mL). Microvascularobservations of the awake and unanesthetized hamster were performed atleast two days after chamber implantation, thus mitigating post-surgicaltrauma. During these investigations, the animals were placed in a tubefrom which the window chamber protrudes to minimize animal movementwithout impeding respiration.

A preparation was considered suitable for experimentation if microscopicexamination of the window chamber met the following criteria: i) nosigns of bleeding and/or edema; ii) systemic mean blood pressure above80 mm Hg; iii) heart rate above 320 beats/minute (Beckman recorder,R611, Spectramed transducer P23XL); iv) systemic hematocrit above 45%(Readacrit® centrifuge, Bectron); and v) number of immobilizedleukocytes and leukocytes flowing with venular endothelial contact lessthan 10% of all passing leukocytes at time point control.

Unless otherwise indicated, the experiments described hereafter werecarried out exclusively in the hamster window preparation. This modelwas selected because it allows observation of the microcirculation forprolonged periods (i.e., several days) in the absence of anesthesia;previously performed microvascular studies indicated that data obtainedfrom anesthetized animals is not representative of the awake condition.The hamster window preparation also presents the tissue being observedin a state that is isolated from the environment in order to obtainrepresentative data.

Intravital Microscopy

Microscopical observations were performed using an intravital microscope(Leitz, Ortholux II) with a 25×SW 0.60 n.a. water immersion objective.The preparation was observed visually with a 10× ocular at a totaloptical magnification of 250×. Contrast enhancement for thetransilluminated image was accomplished by using a blue filter (420 nm),which selectively passes light in the maximum absorption band ofhemoglobin, causing the red blood cells to appear as dark objects in anotherwise gray background. A heat filter was placed in the light pathprior to the condenser.

The microscopic images were viewed by a closed circuit video systemconsisting of two different cameras, a video cassette recorder (SharpXA-2500S) and a monitor (Sony, PVM 1271Q), where total finalmagnification at the monitor was 650×.

Capillary Red Blood Cell Velocity

Capillary red blood cell velocity (CRBCv) was measured using the videodual window technique with a velocity tracing correlator (IPM, model102B). CRBCv for each capillary was measured for a period of 20 secondsin order to obtain an average velocity over the period of observation.All measurements were performed in the same capillaries. Thosecapillaries that had blood flow and which stopped at subsequent timepoints were not included in the statistics with a zero value at the timepoint in which there was no flow; this is because their effect on tissueperfusion index is accounted for by their effect on the functionalcapillary density (FCD), i.e., the number of capillaries in a unit areaobserved to be passing RBCs. CRBCv was measured in one-to-two vesselsper field of observation (10-12 per animal), since not all capillariesin a field are in the same focal plane.

Arteriolar and Venular Diameters

Arteriolar and venular diameters were measured at each time point usingan image shearing monitor (IPM, model 907) during video playback.

Measurement of pO₂ in Microcirculation

Before collection of data, each animal received a slow intravenousinjection of palladium (Pd)-coproporphyrin (Porphyrin Products, Inc.)previously bound to albumin. The concentration used was 30 mg/kg bodyweight. During pO₂ measurements, a xenon strobe arc (EG&G ElectroOptics) with a decay constant of 10 microseconds was flashed at 30 Hzover a selected area. Epi-illumination was only used during pO₂measurements, in order to avoid possible tissue damage which may becaused by the intense illumination. The phosphorescence emission fromthe epi-illuminated area passes through an adjustable slit and a longband pass filter (cut off at 630 mm) before being captured by aphotomultiplier (EMI, 9855B). Slit size was usually kept at 15×100 μm(relative to the actual microscopic field), and it was always positionedalong the length of the vessel.

When interstitial measurements were performed, the slit was positionedparallel to the nearest vessel, at various distances. The signals fromthe photomultiplier were sent to a digital oscilloscope (Tektronix,2430). The oscilloscope averages 200-500 curves, and a single smoothedcurve was then digitized (10 bit resolution) at a rate of 0.5 MHz andstored for later analysis. Each curve was also processed by aspecialized analog processor for the calculation of pO₂.

General Experimental Protocol

Unless otherwise indicated below, the following general exchangetransfusion procedure was utilized in the examples that follow. Thechamber window of the window preparation was implanted at day one. Thechamber was inspected for compliance with inclusion criteria at day 3,and, if satisfactory, carotid artery and jugular vein catheters wereimplanted. The animal was investigated at day 5 for compliance withsystemic and microvascular inclusion criteria, and, if satisfactory, anexchange experiment was started.

Each experiment served as its own control, and all data were relative tothe conditions of the animal at the start of the experiment. Videomicroscopic measurements, systemic hematocrit, heart rate, blood gasses(pO₂, pH, pCO₂) and blood hemoglobin content (this measurement wasinitiated with the experiments involving Hemolink®/dextran and continuedwith the experiments conducted thereafter) were taken at control priorto exchange of blood. Microscopic measurements at control includedcapillary flow velocity and arteriolar and venular diameters.Microvascular pO₂ measurements were not taken at control, since thismeasurement can only be carried out at one time point due to toxicity.Macro and micro data collection at control lasted one hour.

After control measurements were collected, the first exchange wasinitiated. The target was 40% of the original blood mass to be withdrawnand replaced with a blood substitute at the rate of 100 μL/min (theduration of this procedure was 10-20 minutes). At the end of thisprocedure and after an equilibration and stabilization period of tenminutes, micro and macro measurements, described above, were taken (theduration of this procedure was one hour).

A second exchange targeted at extracting 30% of the original volume wasthen instituted, using the procedure described above. Micro and macromeasurements were taken, and, if this was the final exchange target, theanimal was transferred to the pO₂ measurement microscope. The animal wasinjected with the porphyrin compound and intravascular and extravascularpO₂ measurements were made in arterioles, venules and the tissue (theduration of this procedure was one hour).

If the final hematocrit target was in the range of 20%, then a thirdexchange was performed, and microvascular pO₂ was not measured duringthe second exchange. After the third exchange, micro and macromeasurements were made, and the animal was transferred to the pO₂measurement microscope.

Statistical Analysis

Data obtained for each group were analyzed to determine if the changesobserved within groups were statistically significant. The results ofeach group are presented by treating each data point as resulting froman independent experiment. The Mann-Whitney non-parametric test was usedon the normalized means to assess if the changes in the parameters weresignificantly different from control. Results are given in terms ofmedian and interquartile ranges. Changes were deemed statisticallysignificant for p<0.05.

The examples that follow are divided into the following sections:

-   I) Microcirculation Experiments; and II) Clinical Model Experiments.    1. Microcirculation Experiments

EXAMPLE 1 Blood Flow and Hematocrit During Colloid and SalineHemodilution

The experiments of this example were directed at determining the effectof decreasing hematocrit, as a result of hemodilution, on blood flowvelocity. The experiments of this example were conducted on hamstersusing dextran 70 and saline.

The general experimental procedures (e.g., General Experimental Protocoland Capillary Red Blood Cell Velocity) described above were performed.FIG. 2 depicts a plot of flow velocity in the microcirculation as afunction of hematocrit reductions with dextran hemodilution and salinehemodilution. The following designations are used in FIG. 2: i) dextranhemodilution: small circle=mesentery; square=skin; plus sign=muscle; andii) saline hemodilution: large circle=skin fold. The results indicatethat blood flow, as evidenced by the velocity of blood in the vessel ofthe microcirculation, increases as blood is diluted. The increase islinearly related to the decrease of hematocrit, reflecting the fact thatmost of the viscous losses in the circulation occur in themicrocirculation where the relationship between blood viscosity andhematocrit is linear.

The majority of previous studies have shown that the number of RBCs canbe reduced to 25% of the original amount, i.e., a loss of 75% of theoriginal RBC mass, while maintaining circulatory function and flow. Mostfree hemoglobin solutions (e.g., HBOCs) do not show the linear increasein blood flow with the reduction in hematocrit for very low hematocrits,which is evidenced by non-oxygen carrying diluents. These resultsindicate the presence of additional processes in the case of freehemoglobin solutions, such as the arterial wall reactions previouslyalluded to and described in further detail below.

EXAMPLE 2 pO₂ Distribution During Dextran 70 and Hemolink® Hemodilution

The experiments of this example were directed at determining the effectof hemodilution on pO₂ in the microcirculation by the phosphorescencedecay method described above.

Dextran 70 Hemodilution

Measurements of pO₂ were made in 50 μm arterioles and the tissuesurrounding those arterioles. The results were as follows: arteriole pO₂(pO_(2.A))=53 mm Hg; tissue pO₂ (pO_(2.T))=21 mm Hg. The followingequation may then be utilized to calculate K_(A)*, the constantrepresenting the difference in the decrease in the oxygen partialpressure between i) the arterioles and the tissues and ii) the centralarteries and the tissues:K _(A) *=In[(pO _(2.A) −pO _(2.T))/(pO _(2.a) −pO _(2.T))]where pO_(2.a) is the oxygen tension in a central artery. If one assumesa pO_(2.a)=100 mm Hg, then KA*=In [(53-21)/(100-21)]=−0.90.

Table 5 sets forth previously obtained (by the present inventors) pO₂values for various hematocrit (α) levels with dextran 70 hemodilution.The convection diffusion model allows comparison of measured values totheoretical values. Changes in blood viscosity (γ) were not measureddirectly, but were inferred from the change in blood flow velocity inthe microcirculation; the relative viscosity y relates to the viscosityof whole blood (γ=1.0). The oxygen carrying capacity was assumed to bedirectly proportional to hematocrit (i.e., ignoring oxygen carried byplasma). Table 5 summarizes measured and theoretical pO_(2.A) valuesfollowing dextran 70 hemodilution. Predicted values for each level ofhemodilution were obtained by using model results where K_(A)* wasmultiplied by the corresponding γ/α ratio. TABLE 5 pO_(2.A) pO_(2.A)Theor. mm Meas. Wall Grad. pO_(2.T) α γ γ/α Hg mm Hg mm Hg mm Hg 1.01.00 1.0 53 55 21 0.8 0.80 1.0 53 0.6 0.67 1.12 56 55 21 21 0.4 0.571.42 42 54 22 20 0.2* 0.50 2.50 29 37 17 8*Animals do not tolerate this low hematocrit. The viscosity factor γ isdeduced from the effect on velocity.

The results presented in Table 5 indicate that a reduction of hematocritto 60% of the original amount, i.e., a loss of 40% of the original RBCmass, or a hemoglobin concentration (in RBCs) of 9%, does not normallychange tissue oxygenation. This is true in terms of autoregulatoryresponses and in terms of tissue oxygenation. The model predicts thatblood pO₂ in the arterioles would be significantly lower as hematocritis reduced to 40% and 20% of the normal value. However, as the dataexhibit, this does not take place for reductions of 40%, indicating thatthe arterioles elicit a sufficiently strong autoregulatory responseaimed at sustaining pO₂. Further reductions of hematocrit cause animportant decline in tissue pO₂. Moreover, the wall gradient at extremehemodilution is low, reflecting vasodilation needed to respond to lowerarteriolar oxygen tension.

Hemolink® Hemodilution

Hemodilution with Hemolink® was carried out in an analogous manner tothat described above for dextran 70. The results are set forth in Table6. TABLE 6 pO_(2.A) Theor. pO_(2.A) Meas. Wall Grad. pO_(2.T) (Htc)α* γγ/α mm Hg mm Hg mm Hg mm Hg (0.6)0.86 0.65 0.97 61 (0.4)0.80 0.66 0.8959 55 23 17 (0.2)0.73 0.54 0.91 62 53 28  5*α shows the oxygen carrying capacity of the mixture of HemoLink ®(concentration: 10 g/100 mL) and RBCs. The numbers are normalizedrelative to the oxygen carrying capacity of normal blood.

The results in Table 6 indicate that Hemolink® maintained arteriolar pO₂for all levels of hemodilution. Animals tolerated hemodilution to 20% ofthe original RBC mass, which is not the case with dextran hemodilution.Though an understanding of the mechanism is not required in order topractice the present invention, the maintenance of arteriolar pO₂appears to be due to a vasoconstrictor effect that reduces blood flow byabout 25%. This is evidenced by: i) increased vessel wall gradient (asign of vasoconstriction); ii) arteriolar vasoconstriction; and iii) aflow increase due to viscosity effects that is lower than that obtainedwith dextran 70 hemodilution, as evidenced by higher γ values at anygiven level of RBC mass dilution with Hemolink®.

If dilution with Hemolink® were to increase blood flow only according tothe viscosity effect resulting from colloids, one would expect to obtainpO₂ values at the level of 50 μm arterioles that, when calculatedaccording to theoretical predictions, would be approximately 60 mm Hg(for hematocrit=0.4). Though the practice of the present invention doesnot require an understanding of why the values are approximately thesame, the differences between the theoretical figures and the measuredfigures indicate the existence of some sort of arterial wall reaction.The results suggest that there is a vasoconstrictor effect accountingfor decreased blood flow on the order of 25%, since this would be due toa decrease in vessel diameter on the order of 6%. The data obtainedshows that arteriolar diameters decrease to 93% of control forhematocrit 0.4 and to 88% of control for hematocrit 0.2. This level ofvasoconstriction is also evident from the increase in pressure forhematocrit 0.4 (but not different from control for the greater exchangelevel).

The results obtained with Hemolink® indicate that, following anisovolemic reduction of hematocrit from 10% to 40%, tissue oxygenation(in terms of the pO₂ of 50 μm arterioles and tissue to the same level)is sustained at those levels present in normal conditions. Though aprecise understanding of the methodology of this effect is not necessaryin order to practice the present invention, the observed slight increasein blood pressure and vessel wall gradient and decrease in functionalcapillary density may be the direct consequence of autoregulatoryphenomena, i.e., phenomena aimed at maintaining pO₂ in 50 μm arteriolesconstant in the presence of potentially excess oxygen carrying capacitydue to lowered blood viscosity.

Effect of the Results on Blood Substitute Formulations of the PresentInvention

The results or this example indicate that Hemolink®, in its presentformulation, provides too much oxygen and that the viscosity of theresulting blood mixture is too low. While hemodilution with inertcolloids depends on low blood viscosity to maintain oxygen carryingcapacity, the resulting increase in cardiac output may not be adesirable effect in all cases. Therefore, in some embodiments of thepresent invention, Hemolink® and other oxygen-carrying components,especially HBOCs, are formulated in a solution that contains an inertcolloid. In this way, either an increase in viscosity is achieved and/orthe oxygen carrying capacity is decreased, while colloid osmoticpressure and plasma retention are maintained.

EXAMPLE 3 Tissue Oxygenation Resulting from Hemodilution with 50%Hemolink®/50% Dextran 70

The experiments of this example are directed at determining the adequacyof tissue oxygenation following administration of a mixture of Hemolink®and dextran 70.

A mixture of 50% Hemolink® and 50% dextran 70 was prepared, and tissueoxygenation was determined at hematocrit levels of 60% and 40% ofbaseline levels. Hemoglobin concentration in the resulting mixture wasmeasured directly by spectrophotometry. In addition, the number of RBCsand the amount of Hemolink® were measured directly in blood samples.Though testing was initiated using four animals, only two animalssatisfied all criteria for inclusion in an experimental run; the resultsfor the two animals are set forth in Table 7. TABLE 7 pO_(2.A) Meas.Wall Grad. pO_(2.T) Htc/α γ γ/α pO_(2.A) mm Hg mm Hg mm Hg 0.6/0.68 0.640.86 55 0.4/0.54 0.76 1.41 43 51 27 15

When the data in Table 7 is compared with that derived from use ofHemolink® alone (see Table 6), it is observed that the values ofpO_(2.T) (17 mm Hg v. 15 mm Hg, respectively, for hematocrit=0.4) arevery similar; these values are acceptable in practice. Therefore, boththe diluted mixture and Hemolink® itself provide adequate tissueoxygenation, despite the fact that the mixture carries only half as muchoxygen per unit weight as is carried by Hemolink® alone.

EXAMPLE 4 Tissue Oxygenation with Hemolink®, Dextran 70 andHemolink®/Dextran 70 at Hematocrit 0.4

The experiments of this example are directed at determining andcomparing the tissue oxygenation of Hemolink®, Dextran 70, andHemolink®/Dextran 70 (50%/50%) at hematocrit 0.4. These experimentsbuild upon those set forth in the preceding example.

The efficacy of tissue oxygenation following administration of theabove-mentioned compositions was evaluated from information ofarteriolar and venular pO₂, the percent oxygen saturation of hemoglobin,capillary flow velocity (1/γ), and intrinsic oxygen carrying capacity(α). These parameters were determined as previously described, andoxygen extraction by the microcirculation was determined by the methoddiscussed hereafter. The results are set forth below in Table 8(relative numbers are indicated where applicable). TABLE 8 NormalDextran HemoLink ®/ Blood 70 HemoLink ® Dextran Arteriolar pO₂ 53 54 5551 Arteriolar O₂ % sat. 0.84 0.85 0.85 0.81 Venular pO₂ 33 30 20 22Venular O₂ % sat. 0.52 0.50 0.30 0.32 Cap. Velocity 1.0 1.75 1.51 1.32O₂ carrying capacity 1.0 0.40 0.80 .54 Extraction 0.32 0.22 0.50 0.26

The data in Table 8 for oxygen extraction are derived from measurementsof the pO₂ gradients at the vessel wall. This value, in combination withthe value for oxygen carrying capacity normalized to blood=1, gives anindication of the relative amount of oxygen which is lost between thearterial vessel and the tissue for a given level of tissue oxygenation.In the case of normal (i.e., undiluted blood), the figure is 32%. Whenblood is diluted with dextran 70, the figure is 9% (i.e., 22% of 40%);when blood is diluted with Hemolink®, the figure is 40% (50% of 80%);and when blood is diluted with a dextran/Hemolink® mixture, the figureis 14% (26% of 54%).

The results indicate that the dextran/Hemolink® mixture is considerablymore efficient in delivering oxygen to the tissues than is Hemolink®alone. Because the mixture loses much less of its oxygen in moving fromthe arteries to the capillaries than does Hemolink® alone, the mixturehas greater reserves of oxygen available to the tissue for oxygenationpurposes. Therefore, the compositions of the present inventioncomprising a non-oxygen carrying component and an oxygen carryingcomponent provide greater reserves of oxygen for the tissues; thisresult represents an additional, unexpected advantage of thecompositions.

EXAMPLE 5 Wall Gradients with Hemolink® and Hemolink®/Dextran 70 atHematocrit 0.4

Several of the previous examples were directed at the use of the “awakehamster” model to determine i) partial oxygen pressures in arteries,veins and tissue, and ii) blood pressure in normal blood (control) withHemolink® at hematocrit 0.4, and 50:50 dextran:Hemolink® at hematocrit0.4. This example is directed at the determination of wall gradientsusing each of those compositions.

As previously indicated, the vessel wall gradient is inverselyproportional to tissue oxygenation. In this example, wall gradients werederived from the pO₂ measurements in previous studies. The bloodpressure data represents mean arterial blood pressure relative to thecontrol. The results are shown in Table 9. TABLE 9 HemoLink ®/ ParameterControl HemoLink ® Dextran Wall Gradient - Arteriole 17.8 24.3 26.8 (mmHg) Wall Gradient - Venular 10.1 10.8  7.6 (mm Hg) Tissue pO₂ 21.4 17.019.2 Blood Pressure 100% 112% 109%

The data in Table 9 indicate that the Hemolink®/dextran composition iseffectively equivalent to Hemolink® alone when compared for the measuredparameters. Moreover, the results of this example, in conjunction withthe examples set forth above, indicate that the desirable properties ofa blood substitute obtainable by using Hemolink® (and, by extrapolation,other HBOCs) alone are also obtainable with the compositions of thepresent invention (i.e., compositions comprising solutions of an oxygencarrying component in combination with a non-oxygen carrying component).

EXAMPLE 6 Microcirculatory Parameters at Hematocrit of 12-13%

The experiments of this example utilized the previously describedprocedures to assess various microcirculatory parameters followingadministration of several different compositions.

Six different compositions were administered to hamsters in separateexperiments: 1) control (i.e., normal blood); 2) dextran 70 alone; 3)Hemolink® alone; 4) Hemolink® 33%/dextran 66% (by volume); 5) Hemolink®50%/dextran 50%; and 6) L-Name (L-nitrosyl-arginine-monomethyl-ether;commercially available from, e.g., Sigma). A hematocrit of approximately12% of the control was achieved in experiments 3)-5) following threeexchange perfusions. Only two hemodilutions (i.e., two exchangeperfusions) were performed for the experiment with dextran alone(experiment number 3) because the animals do not tolerate threedilutions with this composition. The L-name composition was injectedinto animals (i.e., it was not administered to effect hemodilution).

The resulting data is set forth in Table 10. Referring to Table 10,PaO₂=arterial PO₂; Grad(A)=arteriolar/tissue gradient; andGrad(V)=venular/tissue PO₂ gradient. The data regarding vasoconstrictionis relative to the control (experiment number 1).

The data in Table 10 indicate that hemodilution with thehemoglobin-based oxygen carrier (HBOC) Hemolink® decreased tissue PO₂from approximately 20 to 5 mm Hg. This was accompanied by an increase ofthe arteriolar/tissue PO₂ gradient from about 17 to 28 mm Hg, consistentwith the vasoconstriction previously determined to be caused by thisproduct. When the Hemolink® was mixed with the non-oxygen-carryingplasma expander dextran, tissue PO₂ increased to 13 and 17 mm Hg,respectively, with 33% and 50% mixtures of Hemolink®/dextran. However,in the experiments with the Hemolink®/dextran compositions, thearteriolar/tissue PO₂ gradient remained high, a consequence ofvasoconstriction still being produced by the hemoglobin.

These experiments, in conjunction with some of the results from theprevious examples indicate that if the O₂ availability is increased bythe extracellular location of hemoglobin, then, in order to preventautoregulatory vasoconstriction at the arteriolar level, one or more ofthe following compensations must take place: i) increased viscosity, ii)decreased O₂ carrying capacity, or iii) increased O₂ affinity. TABLE 10PaO₂ Grad(A) Tissue Venular Grad(V) Arteriolar Exp. n Material Hct % BPTorr Torr pO₂Torr pO₂Torr Torr Vasoconstriction Velocity 1 Controlstable 53 17.8 20 33 10 2 Dextran 19 unstable 54 22 20 30 11 none 1.4 3many HemoLink 12 stable 53 28 5 10 4 not done 1.2 4 1 HemoLink 33% 12stable 69 34 13 25 18 0.2 Dextran 66% 5 2 HemoLink 50% 12 stable 73 4117 21 8 0.3 2.6 Dextran 66% 6 many L-Name stable 57 26 21 28 9 not done1.8

EXAMPLE 7 Use of a Composition Comprising Hemolink® andPolyvinylpyrrolidone

The experiments of this example provide evidence that increasedviscosity prevents autoregulatory vasoconstriction at the arteriolarlevel. The microvasculature experiments of this example were performedutilizing a composition comprising Hemolink® and polyvinylpyrrolidone(PVP), 750,000 dalton molecular weight.

Aqueous solutions of i) Hemolink®, ii) 50:50 Hemolink®:dextran molecularweight 70,000 (by volume), and iii) 100:4 Hemolink®:PVP molecular weight750,000 (by volume) were prepared at a total solute concentration, ineach case, of 10 g/100 mL. The compositions were tested in the “awakehamster” model described above. PVP is used experimentally as a plasmaexpander and has also been used in humans for the same purpose; itsprincipal property is that of increasing plasma blood viscosity. The useof PVP substantially increases the viscosity of the solution, to a valueestimated at about 15 centipoise (substantially equivalent to that ofwhole blood).

The animals were subjected to an isovolemic exchange of blood with eachof the compositions to achieve a final hematocrit of 0.20 of control(i.e., 20% of original RBC mass) or an effective hematocrit of about10%. By the procedures previously described, measurements were taken ofthe arterial pressure, wall gradient, blood pressure and tissue oxygen.The results are set forth below in Table 11.

The results in Table 11 indicate that the increased viscosity of theHemolink®:PVP composition significantly lowers the vessel wall gradient,making more oxygen available to the tissue, compared to the other twocompositions. This increased viscosity causes dilation of thevasculature and normalizes the distribution of oxygen in themicrocirculation. Though an understanding of the underlying mechanism isnot required in order to practice the present invention, the mechanismfor vasodilation with compositions of increased viscosity is believed tobe two-fold. First, decreased oxygen delivery of blood due to lowerhemoglobin causes autoregulatory effects analogous to those observedwith the previously described oxygen-carrying compositions comprisingother inert, non-proteinaceous colloids. TABLE 11 Relative pO₂Arterioles Wall Gradient Mean Arterial Blood pO₂ Tissue Hb Content αViscosity γ mm Hg mm Hg FCD Pressure % Normal mm Hg HemoLink ® 0.73 0.5453 28 0.64 −14%   5 HemoLink ® & 0.54 0.76 51 27 0.78 +9% 13 DextranHemoLink ® & 0.51 1.00 46 15 0.45 −3% 16 PVPSecond, increased shear stress at the vessel wall increases release ofendogenous vasodilators such as prostacyclin.

In addition, even though the O₂ capacity of the Hemolink®/PVP mixture islower than that of Hemolink® alone and its viscosity is higher, thearteriolar/tissue PO₂ gradient is reduced, and tissue PO₂ is increasedfrom 5 to 16 mm Hg. These results are consistent with the theoreticalformulation alluded to previously. However, it is believed that themixture of Hemolink® and PVP is not suited to development as a bloodsubstitute, and the functional capillary density is lower than desired.

II. Clinical Model Experiments

EXAMPLE 8 Use of Pentastarch, Hemolink®, and a Mixture Thereof UnderClinical Conditions

This example relates to experiments conducted in vivo using maleSprague-Dawley rats under severe stress. The experiments of this exampleprovide information relevant to the clinical use (e.g., in an operatingtheater environment) of the compositions of the present invention.

Exchange Transfusion

The animals were instrumented 24 hours prior to initiation ofexperiments, and all experiments were conducted in the awake state. Acatheter was placed in the femoral artery and another in the femoralvein. The animal was restrained in an experimental cage. First, anexchange transfusion was performed in which about 50% of the blood ofthe animal was removed and replaced with a test composition; the testcompositions assessed were pentastarch, Hemolink® and aHemolink®/pentastarch mixture (50:50 by volume). A peristaltic pump wasused to simultaneously withdraw blood and infuse one of the testcompositions at a rate of 0.5 mL/min. The duration of the exchange wascalculated to achieve exchange of 50% of the estimated total bloodvolume, based on 65 mL of blood per kg body weight as the standard bloodvolume of the rat.

Mean Arterial Blood Pressure During Exchange

As the exchange transfusions proceeded, mean arterial pressures weremeasured through the catheter, by standard procedures in the art. FIG. 3graphically presents arterial blood pressure prior to and during theexchange transfusion (indicated by the arrow in FIG. 3). Referring toFIG. 3, (▾) represents Hemolink®, (▴) represents pentastarch and (∇)represents the mixture of Hemolink® pentastarch. Using the statisticalanalyses described above, there are no significant differences betweenHemolink® alone and the composition of Hemolink®/pentastarch.

Physiological Status During Hemorrhage

Animals were subjected to a 60% hemorrhage procedure analogous to thatdescribed in the preceding example. More specifically, 60% of the totalblood volume was calculated, using the aforementioned 65 mL/kg estimate.The calculated amount of blood was then removed using a simplifiedexponential protocol similar to that developed by Hannon et al. (“Bloodand Acid-base Status of Conscious Pigs subjected to Fixed-volumeHemorrhage and Resuscitated with Hypertonic Saline Dextran,” CirculatoryShock 32:19-29 [1990]). At the beginning of each 10 minute period of thehemorrhage, blood was removed from an arterial site using a syringe pumprunning at a rate of 0.5 mL/min. The duration of each withdrawal wascalculated so that 60% of the total blood volume was removed over 60minutes.

Mean arterial blood pressure was measured through the catheter, and dataare presented graphically in FIG. 4; in FIG. 4, the symbols depictingeach composition are the same as set forth in FIG. 3. Of note, theanimals transfused with the Hemolink®/pentastarch composition start thebleed with a higher blood pressure, which initially falls quite steeply.Both the Hemolink®/pentastarch composition and Hemolink® alone preservethe blood pressure well during the first 50 minutes.

The hemorrhage test described above represents a relatively severe testmodel. Only about 50% of the animals, even without an exchangetransfusion, survive beyond 120 minutes from the onset of the 60%hemorrhage, and even fewer of those transfused with a test solutionsurvive (data not shown).

Other measurements were also determined during the hemorrhaging,including heart rate (measured from the pressure trace of the meanarterial pressure measurements), and pH, pCO₂, pO₂, lactateaccumulation, and base excess (measured by standard analysis of theblood). The results (not shown) from animals transfused with Hemolink®and those transfused with the Hemolink®/pentastarch composition weresubstantially equivalent with the following exception. TheHemolink®/pentastarch composition resulted in more lactate accumulation,reflecting the fact that this composition carries less oxygen. Lactateaccumulation is a direct reflection of the status of tissue oxygenation;that is, lactate accumulates when tissue is not supplied with sufficientoxygen.

The findings of the experiments of this example indicate that a mixtureof an oxygen-carrying component and a non-oxygen carrying componentprovides similar, if not superior, results to that achieved with anoxygen-carrying component alone.

EXAMPLE 9 Use of Pentastarch, Modified Hemoglobins, and Mixtures ThereofUnder Clinical Conditions

The experiments of this example evaluate two oxygen-carrying components,bovine hemoglobin modified by conjugation with polyethylene glycol(PEGHb or PEG) and αα-Hb, alone and in combination with a thenon-oxygen-carrying component, the plasma expander pentastarch(Pentaspan®; DuPont).

Nature of the Compositions

The properties of several of the compositions used in this example arecompared in Table 12. The PEGHb+pentastarch composition and theαα-Hb+pentastarch composition comprised 50% of each composition byvolume. As indicated in Table 12, both PEGHb and pentastarch have highcolloid osmotic pressure (COP) values, and both have a viscosity thatapproximates that of blood (in the measuring system used, water andpurified hemoglobin have viscosities of 1 centipoise). TABLE 12Hemoglobin Viscosity Solution COP (mm Hg) (g/dL) (centipoise) Blood 26.015.0 4.0 Pentaspan ® 85.0 0.0 4.0 PEGHb 81.3 6.0 3.4 PEGHb + Pentaspan98.0 3.0 3.2

Exchange Transfusion

A 50% isovolemic exchange transfusion was performed in awake rats usingthe procedure described in the preceding example. Table 13 indicates theeffect of the exchange transfusion (±sem) on blood volume, hematocrit,total hemoglobin, and plasma hemoglobin for several of the compositions.TABLE 13 Blood Volume Plasma Hb Solution (mL/kg) Hct (%) Total Hb (g/dL)(g/dL) Controls 56.3 ± 2.5 38.6 ± 0.9 13.8 ± 0.3  0.0 ± 0.0 Pentastarch71.1 ± 5.7 18.4 ± 1.0 6.8 ± 0.4 0.0 ± 0.0 PEGHb 74.0 ± 1.6 15.8 ± 0.47.6 ± 0.1 2.0 ± 0.1 PEGHb + 91.0 ± 3.0 14.8 ± 0.3 5.6 ± 0.2 1.0 ± 0.1PentaspanReferring to Table 13, the decreases in hematocrit and hemoglobinconcentration for the experimental groups indicate that the exchangeprocedure led to significant expansion of the plasma volume in thePEGHb, Pentaspan® and PEGHb+Pentaspan® animals.

Physiological Status During Hemorrhage

Next, the rats were subjected to a 60% hemorrhage over 1 hour; thisprotocol, known to be lethal in approximately 50% of animals, wasperformed as described in Example 8.

In FIGS. 5-10, the following designations apply: pentastarch (▴), αα-Hb(▪), PEG-Hb (●), pentastarch+αα-Hb (□), pentastarch+PEG-Hb (∘), andcontrol animals (♦). FIG. 5 depicts animal survival over a 2 hour periodbeginning with the start of hemorrhage. As indicated by the data in FIG.5, hemodilution with pentastarch alone led to significantly reducedsurvival, while hemodilution with either PEGHb alone orPEGHb+pentastarch led to complete survival; survival followinghemodilution with the compositions comprising αα-Hb was much lower thanwith the compositions containing PEGHb.

FIG. 6A-D graphically depict the acid-base status of control rats (♦)and of rats following exchange transfusion with pentastarch (▴), αα-Hb(▪), PEG-Hb (●), pentastarch+αα-Hb (∇), and pentastarch+PEG-Hb (∘) andafter the initiation of a 60% hemorrhage. FIG. 6A depicts PaO₂, FIG. 6Bdepicts PaCO₂, FIG. 6C depicts arterial pH, and FIG. 6D depicts baseexcess.

FIGS. 6A-D are directed at the animals' acid-base status determined overa 2 hour period from the start of hemorrhage. More specifically, FIG. 6Adepicts PaO₂, FIG. 6B depicts PaCO₂, FIG. 6C depicts arterial pH, andFIG. 6D depicts base excess. As indicated in FIGS. 6A-C, neither thePEGHb nor the PEGHb+pentastarch animals had significant respiratoryalkalosis compared to the pentastarch animals. Moreover, neither thePEGHb nor the PEGHb+pentastarch animals developed significant acidosis,even at the end of the hemorrhage period. Acid base status was wellpreserved in the PEGHb and PEGHb+pentastarch animals (FIG. 6D). Again,neither of the compositions comprising αα-Hb performed as well asPEGHb+pentastarch animals or the pentastarch animals.

FIG. 7 shows the production of lactic acid following administration ofeach of the compositions. As depicted in FIG. 7, generation of lacticacid during the hemorrhage was significantly greater in the αα-Hbanimals (alone and in combination with pentastarch) and the pentastarchanimals than in the other groups. Notably, the controls animals (noprior exchange transfusion) and the PEGHb+pentastarch animals hadapproximately equal minimal rises in lactic acid, even though the totalhemoglobin concentration and hematocrit were significantly less in thePEGHb+pentastarch group. (See Table 13).

FIG. 8A depicts mean arterial blood pressure of control rats (♦) and ofrats following exchange transfusion with pentastarch (▴), PEG-Hb (●),and Pentaspan+PEG-Hb (∘) at time −60 minutes, and after the initiationof a 60% hemorrhage at time 0 minutes. As indicated by the data in FIG.8A, blood pressure did not rise in any of the groups during the exchangetransfusion (i.e., from −60 to 0 minutes), but fell significantly in thecontrols and in the pentastarch animals during hemorrhage (i.e., from 0to 120 minutes). Both the PEGHb and the PEGHb+pentastarch compositions“protected” the animals from hypotension.

FIG. 8B depicts mean arterial blood pressure in control rats (♦), andrats following exchange transfusion with pentastarch (▴, point B), αα-Hb(▪, point B), and pentastarch+αα-Hb (□, point A), and after theinitiation of a 60% hemorrhage (point C). As set forth in FIG. 8B, thecontrol animals and the pentastarch animals maintained mean arterialpressure to a greater extent than the pentastarch+αα-Hb animals.

FIG. 9 and FIG. 10 depict relative cardiac output and systemic vascularresistance, respectively. Cardiac output refers to the amount of bloodpumped by the heart in a unit period of time (e.g., liters per minute);relative cardiac output refers to the cardiac output of the threeexperimental groups relative to the control period (−30 minutes). Asdepicted in FIG. 9, cardiac output was higher in PEGHb andPEGHb+pentastarch compared to the other groups. FIG. 10 indicates thatsystemic vascular resistance remained low in both PEGHb andPEGHb+pentastarch animals relative to the other animals.

The results presented above indicate that the PEGHb+pentastarch mixturewas superior to compositions comprising αα-Hb. In addition, thePEGHb+pentastarch mixture performed similarly to the PEGHb compositionalone. This was true even though the hemoglobin concentration to whichthe animals were exposed and the amount of hemoglobin product used wereless by half with the mixture, offering the advantage of reducing theconcentration of hemoglobin given to patients, thereby reducing bothcost and potential side effects. Though a precise understanding of whythe mixture is effective is not required in order to practice thepresent invention, the effectiveness of PEGHb+pentastarch is thought toresult from its preservation of all four of the previously discussedproperties, namely oncotic pressure, viscosity, oxygen affinity, and lowoxygen capacity. Indeed, the results indicate that compositionscomprising i) an oxygen-carrying component (e.g., a HBOC) with highoncotic pressure, oxygen affinity and viscosity and ii) anon-oxygen-carrying plasma expander with similar oncotic pressure andviscosity provide an optimal blood product.

EXAMPLE 10 Survival Data with Modified Hemoglobins, Non-Oxygen-CarryingComponents, and Compositions Thereof

This example is directed at animal survival using several modifiedhemoglobin products (i.e., oxygen-carrying components),non-oxygen-carrying components, and several mixtures comprising anoxygen-carrying component and a non-oxygen-carrying component.

Experimental Protocol

Generally speaking, the experiments of Examples 10-15 were carried outas described in Example 8. Briefly, male Sprague-Dawley rats wereinstrumented, under anesthesia, 24 hours prior to hemodilution.Instrumentation consisted of cannulation of the femoral artery and veinand exteriorizing the catheters so that the animals had free range intheir cages in the following 24 hours. The experiments were all carriedout in awake animals, loosely constrained to restrict gross movements.Arterial pressure was continuously monitored at one femoral artery.Thereafter, 50% of the estimated blood volume (60 mL/kg) was exchangedwith test material at a rate of 0.5 mL/min. This was performed with aperistaltic pump so that withdrawal and infusion were donesimultaneously at the same rate.

Hemorrhage was initiated after the exchange transfusion; the hemorrhagevolume was calculated to be 60% of the original blood volume. Blood wasremoved using a simple exponential protocol so that the hemorrhage wascomplete after 60 minutes. Specifically, the withdrawal pump was drivenat 0.5 mL/min for decreasing periods of time at the start of each 10minute period for a total of 60 minutes.

Animal Survival

Table 14 summarizes all the materials used in the experiments. Referringto Table 14, it should be noted that the designation “DBBF” refers tohuman hemoglobin crosslinked between the alpha chains (“αα-Hb”); thiswas produced by the United States Army and provided as a gift. Twohemoglobin products modified with polyethylene glycol were tested. PHPHemoglobin is a human-derived product from Apex Bioscience, and PEGHb isa bovine-based product obtained from Enzon, Inc. The two PEG-modifiedhemoglobin products (PHP and PEGHb) gave qualitatively the same results.Though the experiments described hereafter utilize PEGHb, other productscomprising PEG-modified hemoglobin and a non-oxygen-carrying component,including, but not limited to, products comprising PHP, are contemplatedby the present invention. TABLE 14 Materials Raw Oxygen Abbr. NameMaterial Source Mol Wt. COP Viscosity Affinity PS Pentaspan Corn DuPontMerck *250,000  High High None HS Hetastarch Corn Fresenius *480,000 Low undetermined None BOV Bovine Hemoglobin Cow Blood Enzon 64,000 LowLow High DBBF αα-Hemoglobin Human Blood U.S. Army 64,000 Low Low Normalβ82 β82 Hemoglobin Human Blood Hemosol 64,000 Low Low High TM TMHemoglobin Human Blood Hemosol 64,000 Low Low Low HL HemoLink ™ HumanBlood Hemosol 128,000  Low Low High PHP PHP Hemoglobin Human Blood ApexBioscience 105,000  Moderate Moderate Mod High PEG PEG Hb Cow BloodEnzon 118,000  High High High*weight-average molecular weight.

One of the major criteria for an effective blood substitute product isenhanced survival, and Table 15 provides several indices of animalsurvival. Specifically, Table 15 sets forth the mean times to death; thecolumn indicating “initial death” refers to the number of minutes thatelapsed following the initiation of hemorrhage before the death of thefirst animal, and the column indicating “% survival” refers to thenumber of minutes that have elapsed when 50% of the animals haveexpired.

Referring to Table 15, all of the mixture blood products (i.e.,Pentaspan®+Hemolink®; hetastarch+Hemolink®; Pentaspan®+PEGHb; andPentaspan®+DBBF) in Table 15 contained 50% (by volume) oxygen-carryingcomponent and 50% non-oxygen-carrying component. These data show thatall of the modified hemoglobins (regardless of their properties), withthe single exception of hemoglobin modified by conjugation withpolyethylene glycol (PEG), show a diminished survival compared tocontrols or Pentaspan®. Indeed, in studies with a mixture of PEGHb and anon-oxygen-carrying component, most of the animals were still aliveafter the one-hour observation period following hemorrhage.

As indicated in Table 15, of the mixture blood products, onlyPentaspan®+PEGHb performed as well as or better than the controls(control animals underwent no exchange transfusion). Moreover,Pentaspan®+PEGHb was nearly as effective in survival as PEGHb, which issurprising given the fact that the total hemoglobin is less in thePentaspan”+PEGHb animals, and the plasma hemoglobin is onlyapproximately 1 g/dL. The animal survival data with the other mixtureblood products was much less than the control animals. TABLE 15 SurvivalInitial 50% Survival Sample Death (Min) Slope (minutes) Controls 110−0.0247 130.2 PS 96 −0.0325 111.4 HS 38 −0.0237 59.1 DBBF 46 −0.017574.6 TM 41 −0.0559 49.9 B82 40 −0.0383 53.1 HL 39 −0.0289 56.3 PEGHb >120 >120 PS/HL 33 −0.0182 60.5 HS/HL 40 −0.0204 64.5 PS/DBBF 33−0.0491 43.2 PS/PEGHb >120 >120

As previously indicated, blood products comprising pentastarch (e.g.,Pentaspan®) and PEGHb optimize viscosity, oncotic pressure, oxygenaffinity and oxygen capacity. Of the products listed in Table 14, onlyPEGHb has all of these properties. Diluting PEGHb with a differentnon-oxygen-carrying component (e.g., the plasma expander hetastarch)would reduce the resulting blood product's viscosity and oncoticpressure, not change the oxygen affinity, but reduce the oxygencapacity. In contrast, the mixtures resulting from combination ofPEG-modified hemoglobin with pentastarch have viscosity and oncoticpressure values very close to that of PEGHb alone.

The examples that follow compare several different blood productmixtures and solutions and summarize the physiological data generatedfrom each set of experiments. The data indicate that preferredsubstitute blood products incorporate most, if not all, of theabove-mentioned properties (i.e., oncotic pressure, viscosity, oxygenaffinity and oxygen content).

EXAMPLE 11 Conventional Plasma Expanders

This example specifically compares animal survival and physiologicaldata following exchange transfusions and hemorrhage with twoconventional plasma expanders (i.e., non-oxygen-carrying components,hetastarch (HS) and pentastarch (PS) (see Table 14)). The experimentswere performed as described in Example 10.

Product Characteristics

Hetastarch is commercially available from Fresenius, and pentastarch wascommercially obtained from DuPont Merck. Both products comprisehydroxyethyl starch, but pentastarch's low molecular weight (250,000 Davs 480,000 Da) is a result of a lower degree of hydroxyethylsubstitution (0.45 compared to 0.70). This difference results in higheroncotic pressure for pentastarch and its faster enzymatic degradation inthe circulation. Because of its higher oncotic pressure, pentastarch hasa greater plasma expanding capability.

Animal Survival

Overall animal survival for the two groups of test animals (pentastarchand hetastarch) and control animals are set forth in Table 15, supra.The data are consistent with the hemodynamic, oxygen transport, andacid-base data. That is, survival in the pentastarch animals issignificantly longer than that of the hetastarch animals, but both areshorter than the controls.

Hematocrit and Hemoglobin

Tables 16, 17, and 18 indicate hematocrit, total hemoglobin, and plasmahemoglobin, respectively. In Tables 16-18, “n”=the number of animals inthe experiment, “ND”=not determined, “post ET” immediately following theexchange transfusion, and “60 min”=following the 60 minute hemorrhage.TABLE 16 Hematocrit Solution n Baseline Post ET 60 Min. Control 7 38.6 ±0.9  24.8 ± 0.9 PS 4 42.6 ± 1.3 18.4 1.0  15.0 ± 0.7 LHS 2  4.0 ± 2.018.3 1.8  12.7 ± DBBF 6 39.5 ± 0.7 18.5 0.4  13.4 ± 0.6 TM 6 42.4 ± 0.921.8 0.5 *13.9 ± 0.2 B82 4  2.7 ± 1.3 18.3 1.0  14.4 ± 0.6 HL 4 40.7 ±1.2 18.1 1.3  12.2 ± 0.8 PEG 5 40.5 ± 1.2 15.8 0.4  12.9 ± 0.2 Bovine 140.0 22.0 #18.2 PS/DBBF 5 40.3 ± 1.1 22.2 2.3 *17.1 ± 2.0 PS/HL 5 43.3 ±0.9 20.2 0.6  15.0 ± 1.0 HS/HL 4 40.5 ± 0.4 19.0 0.1  13.1 ± 0.3 PS/PEG2 40.2 ± 0.8 14.8 0.4  12.6 ± 0.4*50 minute sample.#30 minute sample.

TABLE 17 Total Hemoglobin Solution n Baseline Post ET 60 Min. Control 713.8 ± 0.3  8.8 ± 0.3 PS 4 15.2 ± 0.4  6.8 ± 0.4  5.4 ± 0.3 HS 2 14.1 ±0.9  6.6 ± 0.5  4.2 ± DBBF 6 14.0 ± 0.3 10.2 ± 0.2  7.2 ± 0.4 TM 6 14.8± 0.3 10.9 ± 0.3 *7.4 0.4 B82 4 14.7 0.4 9.2 0.3  7.5 0.4 HL 5 14.2 0.210.8 0.2  7.8 0.1 PEG 5 14.5 ± 0.6  7.6 0.1  6.4 0.1 Bovine 1 14.0 9.9#8 PS/DBBF 5 13.9 ± 0.4 9.1 0.7 *7.1 ± 0.3 PS/HL 5 13.9 ± 1.5  8.8 ± 0.4 6.0 ± 0.1 HS/HL 4 14.4 ± 0.2  8.6 ± 0.1  6.0 ± 0.1 PS/PEG 2 14.0 ± 0.2 5.6 0.2  5.0 0.2*50 minute sample.#30 minute sample.

TABLE 18 Plasma Hemoglobin Solution n Baseline Post ET 60 Min. Control 6No 3.9 ± 0.1  2.3 ± 0.1 PS 6 No 3.7 0.2 *2.2 0.1 HS 4 No 3.6 0.2  2.40.1 DBBF 4 No 4.8 ± 0.1  2.6 ± 0.3 TM 5 No 1.9 0.1  1.5 0.1 B82 5 No 1.6± 0.4 *1.3 ± 0.2 PS/HL 5 No 2.1 ± 0.4  1.1 ± 0.3 PS/PEG 2 No 1.0 0.0 0.8 0.0 Bovine 1 No 2.5 #1.9

The data in Table 16 indicate that both pentastarch and hetastarchhemodilute to a similar degree, as measured by post-exchange hematocrit.However, the hematocrit in the hetastarch animals was significantlylower than in the pentastarch animals after the 60% hemorrhage.Similarly, Table 17 shows that the total hemoglobins were similar inboth groups of animals after hemodilution, but the hetastarch animalshad significantly lower hemoglobin after the hemorrhage.

Hemodynamics

Compared to controls, both pentastarch- and hetastarch-hemodilutedgroups dropped their blood pressure very rapidly after start of thehemorrhage (data not shown). Recovery was faster in the pentastarchanimals and was sustained better than in the hetastarch group, but bothhave significantly lower blood pressure than the controls after thefirst 40 minutes of hemorrhage.

Both hetastarch and pentastarch groups increased their heart rates inresponse to the volume loss, but the rise in the hetastarch group wasmore abrupt than in the pentastarch or control groups (data not shown).Though the practice of the present invention does not require anunderstanding of this effect, it is most likely due to the moresignificant plasma volume expansion expected after exchanging with thehyper-oncotic pentastarch. Both test groups raised their blood pressuresooner than the controls during the hemorrhage, probably because of thesignificantly lower hemoglobin and hematocrit in the exchanged animals.

The parameter dP/dt represents the maximum positive slope of the pulsepressure contour. This parameter is proportional to the onset of thesystolic contraction, and is therefore a reflection of the strength, orinotropic action of the heart. In the control animals, dP/dt rose afteronset of hemorrhage, as the heart attempts to increase its output. ThedP/dt value rose in all three groups, but sooner in the hetastarch groupcompared to pentastarch group and controls (data not shown). Theincrease in dP/dt in the pentastarch group was actually very similar tothat seen in the controls, suggesting that the plasma volume expansionof the pentastarch animals was beneficial.

Ventilation

Ventilation is reflected by PO₂ and PCO₂ measurements. The rise in PO₂and fall in PCO₂ (data not shown) was more pronounced in the hetastarchanimals compared to the pentastarch animals, but both were moresignificant than in the controls. This is a reflection of compromise inoxygen delivery during hemorrhage in the rankhetastarch>pentastarch>Control. Though the practice of the presentinvention does not require an understanding of the mechanism, it isprobable that both starch products reduce the hemoglobin significantlycompared to the control, explaining why both seem to stress the animalsmore than the controls. Of pentastarch and hetastarch, however,pentastarch affords better compensation to hemorrhage, most likelybecause of its better plasma expanding ability.

Acid-Base Balance and Lactic Acid

Regarding pH and base excess, the most significant compromise duringhemorrhage was seen in the hetastarch animals, which exhibited dramaticdrops in pH and base excess (data not shown). The pentastarch animalswere slightly more compromised compared to controls. It is noteworthythat the controls actually seemed to compensate fairly adequately to the60% hemorrhage; specifically, although PCO₂ fell and base excess becamemore negative, the animals were able to maintain their pH essentiallyconstant.

Lactic acid is an accurate indicator of tissue oxygenation. The lacticacid accumulation in the hetastarch animals was significantly greaterthan in the pentastarch animals, and both groups accumulated more lacticacid than the controls (data now shown). Of note, the lactic acid levelplateaued in the controls, suggesting that the rate of production andclearance is equal, another indication of adequate compensation to thehemorrhage.

The results of this example show that in theexchange-transfusion/hemorrhage model utilized, all of the controlanimals were dead by approximately 130 minutes after start of thehemorrhage. Thus, any perturbation in the oxygen transport system wasreflected in a number of measured variables. The results indicate thatneither pentastarch or hetastarch was able to compensate for loss ofhalf of the circulating blood volume. However, comparison of the twoplasma expanders reveals that pentastarch is clearly superior tohetastarch. Though the rationale for this finding is not required inorder to practice the invention, it is believed to be due to the higheroncotic pressure of pentastarch, which thus affords more significantplasma volume expansion in the pentastarch animals compared to thehetastarch group.

EXAMPLE 12 Blood Product Mixtures of Pentastarch and DBBF

This example specifically compares animal survival and physiologicaldata following exchange transfusions and hemorrhage with a blood productmixture (50:50) of pentastarch and DBBF (αα-Hb). The experiments wereperformed as described in Example 10.

Animal Survival

Animal survival of the control animals, pentastarch (PS) alone animals,DBBF (αα hemoglobin) alone animals, and animals administered apentastarch+αα-Hb mixture is shown in FIG. 11. Referring to FIG. 11, (▴)represents pentastarch, (▪) represents αα-Hb, and (□) representspentastarch+αα-Hb. As indicated in FIG. 11, survival of the αα-Hbanimals is significantly worse than either the controls or thepentastarch animals, and a mixture of 50/50 αα-Hb and pentastarch iseven worse. It should also be noted that there was no obviousrelationship between survival and hematocrit (see Table 16, supra) orhemoglobin (see Table 17, supra), so survival does not appear to be alinear function of the oxygen carried in the blood.

Mean Arterial Pressure

Mean arterial pressure rose in the PS/αα-Hb animals and the αα-Hbanimals (data not shown). Moreover, even though hemoglobin dose was halfin the PS/αα-Hb animals, the magnitude of the blood pressure rise wasthe same. Thus, the presence of PS did not attenuate thehemoglobin-induced hypertension of approximately 20 mm Hg. The fall inblood pressure, however, after starting the hemorrhage, was more abruptin the PS/αα-Hb animals than in any of the other 3 groups. The recoverywas somewhat faster, possibly due to the plasma expansion afforded bythe presence of pentastarch. Nevertheless, when blood pressure began tofall terminally, it fell very fast, and animals rapidly died. Thus, therise in blood pressure resulting from the presence of αα-Hb hemoglobindoes not appear to confer any advantage, and the presence of PS does notattenuate this effect.

Heart Rate

In control animals, heart rate gradually increased after start of thehemorrhage (data not shown). Though the present invention does notrequire an understanding of the underlying mechanism of this effect, itis most likely due to loss of intravascular volume. This interpretationis supported by the somewhat lower heart rate response seen in thepentastarch animals who, in spite of a lower hemoglobin concentration,did not raise their heart rate to the same degree (data not shown). Adifferent pattern of heart rate response was seen in the αα-Hb animals;more specifically, there was an immediate drop in heart rate afterstarting the exchange transfusion, followed by a gradual rise afterstarting the hemorrhage (data not shown). The drop cannot be explainedby volume changes, since a contraction of the plasma volume would beexpected to raise the heart rate, not lower it. More likely, this adirect chronotropic effect on the myocardium. Of note, this depressanteffect is lessened when the αα-Hb is diluted with pentastarch (data notshown). The PS/αα-Hb animals exhibited a brisk rise in heart rate afterstarting hemorrhage, rapidly reaching approximately 500/min, a rate notreached in the other groups until a later time. Thus, the PS/αα-Hbmixture did not seem to offer any advantage over αα-Hb alone.

dP/dt

As previously set forth, the dP/dt is the maximum positive slope of thepulse pressure contour. This parameter is proportional to the onset ofthe systolic contraction, and is therefore a reflection of the strength,or inotropic action of the heart. In the control animals, dP/dt roseafter onset of hemorrhage (data not shown). The pentastarch animalsshowed the same pattern, although the magnitude of the response wasless, presumably because these animals had a somewhat increased vascularvolume compared to the controls at the onset of hemorrhage. The αα-Hbanimals never increased their dP/dt (data not shown); in fact, the valuedropped rapidly after the onset of hemorrhage, suggesting that one ofthe normal compensatory mechanisms is disordered. The same observationwas made in the PS/αα-Hb animals, even though they were expected to havea somewhat greater vascular volume than the αα-Hb animals by virtue ofthe presence of oncotically-active pentastarch.

Ventilation

When oxygen transport is diminished, either because of anemia orhypoxia, a normal physiologic response is to hyperventilate. The resultof hyperventilation is a drop in PCO₂, since the elimination of CO₂ bythe lung is a direct function of ventilation. A reciprocal effect isincreased PO₂, again, because of the greater minute volume of gas beingexchanged by the lung. In the control animals, PCO₂ dropped after theonset of hemorrhage (data not shown); by comparison, the pentastarchanimals also lowered their PCO₂ (data not shown), but the effectpersisted for a longer period of time and appeared to be morepronounced, probably as a result of the lower hemoglobin concentrationin the pentastarch animals compared to the controls. (See Table 17). ThePCO₂ drop was significantly greater in the αα-Hb animals, and stillgreater in the PS/αα-Hb animals. Comparison of the αα-Hb and PS/αα-Hbanimals is interesting, since the former has a higher total hemoglobinconcentration, but a lower blood volume. Thus, the addition of PS to theαα-Hb did not confer any advantage on the animals and, in fact, appearsto have induced greater hyperventilation.

The PO₂ changes are the mirror image of the PCO₂ response; the greatestrise in PO₂ (and drop in PCO₂) was seen in the αα-Hb and PS/αα-Hbanimals, while the controls and pentastarch animals had the smallestincrease in PO₂ (data not shown). Thus, the data are consistent with thebelief that reduced oxygen delivery leads to hyperventilation, and thedegree of hyperventilation correlates with overall survival.

Acid-Base Status

As hemorrhage progresses and the delivery of oxygen to tissues becomescompromised, lactic acid is produced and pH drops. For the controlanimals, pH was maintained nearly constant as hemorrhage progressed.Another index of the degree of compensation is the base excess, which isdefined as the amount of base that would be required to return plasma pHto 7.4 in the presence of a PCO₂ of 40 Torr. In the case of both thecontrols and PS animals, base excess was not significantly changed frombaseline (data not shown). In contrast, αα-Hb and, especially, PS/αα-Hbproduced a marked drop in pH which is not compensated by the briskhyperventilation (data not shown), and the result was a dramatic drop inbase excess (i.e., a “base deficit” results). By usual clinicalstandards, a base excess of −10 mEq/L or less is indicative of poorrecovery from hemorrhagic shock.

Finally, lactic acid is a direct measure of the degree of insufficientdelivery of oxygen to tissues (i.e., the “oxygen debt”). Theaccumulation of lactic acid was very significant in both the αα-Hb andPS/αα-Hb animals, the latter rising even more sharply than the former(data not shown). It is also of interest that in the controls andpentastarch animals, there was a rather more modest rise in lactatewhich then seemed to plateau, as the animals' compensatory mechanisms(increased cardiac output, ventilation) seemed to compensate for theblood loss. However, the continued linear rise of lactic acid in boththe αα-Hb and PS/αα-Hb animals indicated progressive, uncontrolledtissue acidosis.

The results discussed above indicate that the use of blood productmixtures comprising αα-Hb as the oxygen-carrying component, even thoughit provides some plasma hemoglobin, rendered the animals in a morevulnerable position with regard to hemorrhage than either the controlsor the animals hemodiluted with pentastarch. The addition of pentastarchto αα-Hb did not compensate for the detrimental effects of αα-Hb and, infact, worsened oxygen delivery, acidosis and overall survival.

EXAMPLE 13 Blood Product Mixtures of Hemolink®/Pentastarch andHemolink®/Hetastarch

Example 8 compared the effects of pentastarch, Hemolink®, and a mixturethereof. This example compares a mixture of Hemolink®/pentastarch with amixture of Hemolink® and another non-oxygen-carrying component,hetastarch. The experiments of this example, performed as described inExample 10, specifically compare animal survival and physiological datafollowing exchange transfusions and hemorrhage.

As previously indicated, Hemolink® (Hemosol) is a polymerized humanhemoglobin that has a mean molecular weight of approximately 128,000 Da.Since Hemolink® is a polymerized product, an array of molecular sizes ispresent in the final product. When tested alone, hemodilution withHemolink® did not perform as well as pentastarch, and animals diedsooner than those in the control or pentastarch groups. (See, e.g.,Example 8). In view of the surprising and positive results with amixture of pentastarch and PEGHb, additional experiments involvingmixtures (50/50) of Hemolink® and a non-oxygen-carrying components(hetastarch or pentastarch) were performed in this example.

Animal Survival

As shown in FIG. 12, exchange transfusion with Hemolink® alone reducedsurvival from a 60% hemorrhage. More specifically, FIG. 12 depictsanimal survival following exchange transfusion with hetastarch (x),Hemolink® (▾), Hemolink®+pentastarch (∇), and hetastarch+Hemolink® (⋄)and after the initiation of a 60% hemorrhage.

The post-exchange hematocrit in the Hemolink® animals (Table 16, supra)was about half that of controls, and slightly lower than thepentastarch, DBBF (αα-Hb), or PS/DBBF animals. However plasmahemoglobins were slightly higher in the Hemolink® animals than in theseother groups (Table 16, supra). FIG. 12 shows that no combination ofHemolink® and either pentastarch or hetastarch was as effective (asmeasured by short-term survival) as the control animals. However, incontrast to the situation with DBBF and pentastarch described in Example12, dilution of Hemolink® with either pentastarch or hetastarch did notworsen survival.

Mean Arterial Pressure

Exchange transfusion with Hemolink® raised mean arterial blood pressureslightly (data not shown), but not as significantly as DBBF (αα-Hb)(Example 12). When the arterial hemorrhage was begun, blood pressure inall four groups (i.e., Hemolink® alone; Hemolink®/pentastarch;Hemolink®/hetastarch; and control) of animals fell abruptly (data notshown). The degree of initial fall in blood pressure was greatest in theHemolink®/hetastarch group (120 to 50 mm Hg) compared to 110 to 80 mm Hgfor the controls, 120 to 90 mm Hg for the Hemolink®/pentastarch animals,and 120 to 80 mm Hg for the Hemolink® alone animals. Thus, as judged bythe fall in blood pressure and overall survival, theHemolink®/hetastarch animals, Hemolink®, and Hemolink®/pentaspan animals(in that order) all seemed to be worse than the controls. Nevertheless,overall survival for the Hemolink®/hetastarch and Hemolink®/pentaspananimals was not different (Table 15, supra) and only marginally betterthan the Hemolink® alone animals.

Heart Rate

The Hemolink® and Hemolink®/pentaspan animals both raised their heartrates in response to the hemorrhage, but the rise was earlier andsteeper than in the controls (data not shown); this indicates lesscardiovascular stability in the exchange-transfused animals compared tothe controls. Surprisingly, the Hemolink®/hetastarch animals droppedheart rate abruptly after starting the hemorrhage; this abnormalresponse might have indicated severe compromise in these animalscompared to the other groups.

dP/dt

An increase in dP/dt was observed in the Hemolink® animals afterexchange transfusion compared to the controls (data not shown),indicating that the exchange by itself conferred instability on thecardiovascular system. The pentastarch/Hemolink® animals demonstratedlittle, if any, increase in dP/dt, whereas the response in thehetastarch/Hemolink® animals was striking, increasing abruptly afterinitiating the hemorrhage, reaching a peak value of nearly 2000 mmHg/sec, and then rapidly falling as animals became severely compromisedand died (data not shown).

Ventilation and Acid Base Status

The rise in PO₂ and fall in PCO₂ observed in each of the threeexperimental groups was greater than the control values, but nodistinction can be made between those groups (data not shown).

All experimental groups demonstrates lower pH during the hemorrhage thanthe control group. The acid-base disturbance was more clearly shown inthe base excess, as all three experimental groups become severelyacidotic (negative base excess) beginning abruptly after start of thehemorrhage. Finally, lactic acid increase was very significant in allthree experimental groups, again confirming the presence of severeacidosis (data not shown).

Previously it was shown that Hemolink® did not perform as well as thecontrols or as well as pentastarch alone; moreover, Hemolink® alone andhetastarch alone performed comparably, but neither afforded as muchprotection as pentastarch alone. As set forth in this example, attemptsto improve the performance of Hemolink® by mixing it with eitherpentastarch or hetastarch did not improve the results.

EXAMPLE 14 Blood Product Mixtures of TM Hemoglobin/Pentastarch

This example specifically compares animal survival and physiologicaldata following exchange transfusions and hemorrhage with a blood productmixture (50:50) of pentastarch and TM hemoglobin; trimesic acid (TM) isused to crosslink hemoglobin. The experiments were performed asdescribed in Example 10.

Animal Survival

TM hemoglobin (Hemosol) is a human-hemoglobin derived product ofmolecular weight approximately 64,000 Da. It has a relatively low oxygenaffinity (P₅₀ about 35 Torr). Studies using TM hemoglobin alone were notsignificantly different from those with DBBF (αα-Hb) alone. (See Example12). All animals that received TM hemoglobin alone or in combinationwith pentastarch died within 60 minutes after start of hemorrhage (onlyone animal was tested using a mixture of TM hemoglobin and pentastarch,and it died at 60 minutes following initiation of hemorrhage).

Mean Arterial Pressure, Heart Rate and dP/dt

Exchange transfusion resulted in a moderate rise in mean arterial bloodpressure of the single animal tested using TM hemoglobin/pentastarch.Pressure abruptly fell after start of the hemorrhage, but then recoveredrather quickly; however as the hemorrhage progressed, when the meanarterial pressure began to fall again, the animal died very suddenly(data not shown).

There was a slight fall in heart rate after the exchange transfusionwith either TM hemoglobin or the TM hemoglobin/pentastarch mixture.After a delay of about 20 minutes, the heart rate rose during hemorrhagein both groups.

Regarding the dP/dt, in contrast to many of the other hemoglobinpreparations, TM hemoglobin/pentastarch or TM hemoglobin alone did notlead to an increase in dP/dt. Rather, a steady fall occurred startingafter the hemorrhage was initiated (data not shown).

Ventilation and Acid-Base Status

As noted in previous examples, PO₂ and PCO₂ change in mirror image,reflecting the hyperventilation that accompanies diminished oxygentransport as hemorrhage progresses. The rise in PO₂ and fall in PCO₂observed in both of the experimental groups was greater than the controlvalues (no distinction can be made between those groups; data notshown).

In regards to arterial pH acid and base, both experimental groupsdemonstrated lower pH during the hemorrhage than the control group; baseexcess determinations showed that both experimental groups becameseverely acidotic (negative base excess) beginning abruptly after startof the hemorrhage (data not shown). Finally, lactic acid increase wasvery significant in both experimental groups (data not shown), againconfirming the presence of severe acidosis.

As previously indicated (see Table 15), TM hemoglobin did not perform aswell as the controls or as well as pentastarch. TM hemoglobin andpentastarch/TM hemoglobin performed comparably, but neither afforded asmuch protection as pentastarch alone. The attempts to improve theperformance of TM hemoglobin by mixing it with pentastarch, reported inthis example, did not improve the results. TM hemoglobin has a low O₂affinity compared to other hemoglobin derivatives studied, and theresults reported above indicate that this low affinity did not conferadvantage over other cross-linked hemoglobins whose other physicalproperties are the same (e.g., DBBF).

EXAMPLE 15 Modified Hemoglobins

As set forth above, mixtures of polyethylene glycol-modified bovinehemoglobin and pentastarch lead to increased animal survival whencompared to mixtures comprising other non-oxygen-carrying components. Inorder to determine whether these results were due to the mixture or tothe bovine hemoglobin itself, an experiment was performed evaluatingpurified bovine hemoglobin. In addition, experiments were performed withβ82 Hemoglobin, a product which has a high oxygen affinity, to determinewhether this product alone might be superior to the mixtures of anoxygen-carrying component and a non-oxygen-carrying componentcontemplated for use with the present invention. As with the previousexamples, the experiments of this example specifically compare animalsurvival and physiological data following exchange transfusions andhemorrhage using the experimental protocol described in Example 10.

Bovine Hemoglobin

Briefly, when the animal was exchange-transfused with bovine hemoglobin,there was only a transient rise in mean arterial blood pressure,followed by a steady fall (data not shown). When the hemorrhage started,mean arterial pressure fell precipitously, and the animal diedapproximately 30 minutes after start of the hemorrhage (data not shown).

The heart rate in this animal did not rise significantly when hemorrhagestarted but there was a modest rise terminally, a few minutes before theanimal died. The dP/dt remained constant, in contrast to controls inwhich this parameter always rose in response to hemorrhage. Finally,regarding pH and acid-base status, the animal severely hyperventilated,as indicated by a rise in PO₂ and a drop in PCO₂. Accordingly, there wasa very precipitous fall in pH and base excess and a sharp rise in lacticacid (data not shown).

β82 Hemoglobin

β82 Hemoglobin (Hemosol) is a derivative of human hemoglobin that iscrosslinked between the β chains (in contrast to DBBF [αα-Hb]). Thisproduct has high oxygen affinity, but low viscosity and oncoticpressure.

When animals were exchange-transfused with β82 Hemoglobin, there was avery transient, but pronounced, rise in blood pressure; the magnitude ofthe rise was approximately the same as that seen with αα-Hb, but themean arterial pressure rapidly returned to the pre-infusion level (datanot shown). When hemorrhage began, blood pressure rapidly fell, andanimals died by approximately 70 minutes. Thus, overall survival was notbetter than αα-Hb hemoglobin, and less than either the controls or thepentastarch animals.

Heart rate did not rise significantly either after exchange or afterhemorrhage, nor did dP/dt. The animals did have pronouncedhyperventilation (increase in PO₂ and fall in PCO₂). Severe acidosis wasshown by a dramatic drop in pH, base excess, and rise in lactic acid(data not shown).

Thus, the experiments with the modified hemoglobin products of thisexample did not lead to superior results than those obtained whenmixtures of pentaspan and PEGHb were employed.

EXAMPLE 16 Evaluation of Various Hemoglobin Solutions

In this Example, three hemoglobin solutions were evaluated (See, Table19), including: 1) Purified human hemoglobin A₀ (Hb-A₀); 2)αα-hemoglobin, human hemoglobin cross-linked with bis(3,5-dibromosalicyl)fumarate; 3) PEG-hemoglobin, bovine hemoglobin surface-modifiedwith polyethylene glycol. The PEG units have a molecular weight of 5,000Da.

Human red blood cells were drawn from healthy volunteers into heparinanticoagulant, washed 3 times in 0.9% NaCl by gentle centrifugation, andresuspended in 0.1 M Bis-tris Cl buffer, pH 7.4. The hemoglobinconcentration of all solutions and red cell suspensions wasapproximately 3 mM (heme). The methemoglobin was always less than 2-4%of total hemoglobin. The test solutions were equilibrated to theappropriate gas concentrations and 37° C. using a tonometer (e.g., model2000, Instrumentation Laboratories, Lexington, Mass.). Human serumalbumin (HSA) was purchased commercially.

The test methods used included the following protocols, the results ofwhich are shown in Table 19. While this Example provides methods todetermine various characteristics of a test preparation, it is notintended that the present invention be limited to these particularprotocols. Indeed, those of skill in the art know additional methodsthat would be suitable for making these determinations.

Oxygen Equilibrium Binding Curves:

Cell-free hemoglobin-oxygen equilibrium curves were measured by couplingdiode array spectrophotometry with enzymatic deoxygenation ofoxyhemoglobin solutions (Vandegriff et al., Anal. Biochem., 256:107-116[1998]). The protocatechuic acid (PCA)/protocatechuic acid3,4-dioxygenase (PCD) enzyme system consumes one mole of O₂ for eachmole of PCA converted to product.

Reactions were carried out 0.1 M bis-Tris propane (Sigma), 0.1 M Cl⁻,and 1 mM EDTA at pH 7.4 and 37° C. Hemoglobin samples were diluted to aconcentration of approximately 60 μM (in heme) in air-equilibrated,temperature-equilibrated buffer containing a small amount of catalase(e.g., 0.2 to 0.5 μM). The final hemoglobin concentration was determinedby the extinction coefficient at 523 nm (ε₅₂₃=7.12⁻¹ mM). Substrate(PCA) was added at a concentration of 1 mM. A volume of this reactionsolution was used to completely fill the reaction cell to eliminate anygas phase present prior to addition of enzyme. The cuvette was sealedusing a gas-tight teflon stopper fitted with a micro-oxygen electrode(Microelectrodes, Inc., Londonderry, N.H.) inserted through an o-ringimbedded in the stopper. The electrode was immersed in the solution to aposition just above the light path of a Milton Roy 3000 diode arrayspectrophotometer (SLM Instruments, Inc., Urbana, Ill.). The temperaturewas controlled using a Peltier controller in the reaction cell holder,and the solution was mixed using a micro-stir bar spun by a stirringmotor in the reaction cell holder. The deoxygenation reaction wasinitiated by addition of enzyme (PCD) (0.05 to 0.1 units/ml).

The spectral change of hemoglobin during enzymatic deoxygenation wasmeasured in the visible range from 480 to 650 nm at every ˜0.35 nm.Polarographic determination of PO₂ was measured using a Clark-typeoxygen electrode, giving a voltage change in proportion to the change inoxygen concentration. The electrode was calibrated each day by immersingthe electrode in water bubbled either with air to determine the 100%-airvoltage or with pure N₂ to set the zero point. During the hemoglobindesaturation reaction, voltages from the O₂ electrode were collected ata sampling rate of 10 Hz.

The spectral and O₂-electrode-voltage data were converted into files foranalysis using the MATLAB technical-computing program (The Mathworks,Natick, Mass.). The voltage output from the O₂ electrode was convertedto mm Hg based on the barometric pressure and the water vapor pressureat the temperature of the experiment. An average PO₂ value wascalculated from 50 data points during each 5-second interval thatcorresponds in time to the collection of each spectrum.

The spectral matrix was analyzed using a multicomponent decompositionalgorithm. The program returns the fractions of each base spectrum(oxy-, deoxy-, and methemoglobin) which combined from the measuredspectrum being evaluated. Fractional saturation was calculated as theratio of oxyhemoglobin to the total of oxy-plus deoxyhemoglobin. Fittedvalues for the Adair constants a₁-a₄) were determined by least-squaresanalysis with uniform weighting. Values for P50 and the Hillcoefficient(n) were calculated from the fitted Adair constants (i.e.,the values shown in Table 19).

Oxygen equilibrium curves for red blood cell suspensions were measuredat pH 7.4 and 37° C. by the gas exchange method using a Hemox-Analyzer®(TCS Medical Products, Huntingdon Valley, Pa.).

COP

COP was measured using a Wescor 4420 colloid osmometer (Logan, Utah)with a 30,000 molecular weight cut-off membrane. The osmometer wascalibrated prior to measurement of each hemoglobin sample with 5%albumin as recommended by the manufacturer. Measurements were performedat room temperature, which ranged from 20-23° C. Values reported inTable 19 are for hemoglobin concentrations of 5 g/dl.

Viscosity

Viscosity measurements are performed using a capillary viscometer(Reinhardt, 1984). The device uses the Hagen-Poiseuille law as itsoperating principle which defines flow (Q) in terms of capillary radius(r), pressure change along the capillary (dP/dx) and viscosity (η).Q=(πr ⁴ dP)/(8 ηdx)

This expression can be separated into two components, the shear stress(L is the capillary length) and the shear rate, where the shear stressand rate are:

-   Shear Stress=(r/2ΔL)ΔP-   Shear Rate=(4/πr³)Q-   Viscosity η=Shear Stress/Shear Rate

Based on the geometry of the capillary, all parameters were known,except ΔP and Q. Thus, these were the two variables measured. Fluid wasplaced in the syringe pump (Harvard Apparatus, model 975, S. Natick,Ma.) and flow started. A differential pressure transducer (ValidyneEngineering, model MP-45-14, Northridge, Calif.) was connected to theends of a 10 cm glass capillary tube with an inside diameter of 508 μm(Vitro Dynamics, Rockway, N.J.) through a T valve. As fluid was driventhrough the tube, the transducer sensed the pressure at each T valvepoint. The transducer was arranged so that the output is the ΔP betweenthe two T valve points. The signal was amplified (Validyne model CD12)and recorded on a strip chart.

Flow (Q) was measured by use of a calibrated flow tube. Viscosity wascalculated from ΔP and Q. The capillary viscometer was both staticallyand dynamically calibrated, while the pressure transducer was calibratedstatically with a head pressure of saline; a dynamic calibration wasaccomplished with water. The solutions were heated to 37° C. and placedin the viscometer. Measurements in the example used a shear rate of 160s⁻¹. The values reported in Table 19 represent the measurements forhemoglobin concentrations of 5 g/dl. TABLE 19 Properties Of The TestSolutions RBC Ao PEG-Hb αα-Hb a₁ 1.48 × 10⁻² 4.01 ± 0.82 × 10⁻² 1.47 ±0.39 × 10⁻¹ 2.22 ± 0.26 × 10⁻² a₂ 8.53 × 10⁻⁴ 1.74 ± 0.44 × 10⁻³ 4.27 ±0.20 × 10⁻² 9.51 ± 0.19 × 10⁻⁴ a₃ 4.95 × 10⁻⁸ 5.95 ± 5.95 × 10⁻¹³ 2.43 ±1.91 × 10⁻⁴ 1.34 ± 0.69 × 10⁻¹¹ a₄ 1.07 × 10⁻⁶ 2.48 ± 0.57 × 10⁻⁵ 1.48 ±0.13 × 10⁻⁴ 1.05 ± 0.13 × 10⁻⁶ P50 (mmHg) 32.8 15.1 10.2 33.8 n  2.59 2.97  1.38  2.43 viscosity  1.4  0.9  3.4  0.9 (cp) (5 g/dl) COP — 1479 11 (mm Hg) (5 g/dl) Radius (nm) —  2.7⁽¹⁾ 14.1⁽¹⁾  3.1⁽¹⁾⁽¹⁾Vandegriff et al., Biophys. Chem., 69: 23-30 [1997].Artificial Capillary Experiments

Exit PO₂ values versus residence times are shown in FIG. 15. At anygiven flow rate, the lowest exit PO₂ value is seen for Hb-A₀ followed byPEG-Hb, αα-Hb, and RBCs with the highest exit PO₂ values. The finalsaturation of hemoglobin in the artificial capillary (FIG. 6) wascalculated from the Adair constants given in Table 19. PEG-Hb showed theleast desaturation over time at any flow rate. This was closelyparalleled by the RBC profile. Hb-A₀ and αα-Hb both showed much greaterdegrees of desaturation.

The finite element analysis adjusts values for the lumped diffusionparameter, K*, until the exit PO₂ equals the experimental value. Thefinal fitted values for K* as a function of residence time are shown inFIG. 17. PEG-Hb and RBCs gave similar values for K* from 900-1200μM/min/Torr. The K* values for Hb-A₀ and αα-Hb are higher than for RBCsbecause of the absence of intraluminal resistances for cell-freesolutions. This effect is negated in the cell-free PEG-Hb solution,which has a K* value equal to that for RBCs at the fastest flow rate andwhich is only slightly higher than RBCs at the slowest flow rate. Thisis due to at least two physical properties of the PEG-Hb solutions (SeeEquation 2, above): (1) its higher viscosity compared with thetetrameric solutions, due to its larger molecular size; and (2) its highO₂ affinity.

Animal Experiments

Male Sprague-Dawley rats (210-350 g, Charles River Labs) wereanesthetized with 250 μl of a mixture of ketamine (71 mg/ml),acepromazine (2.85 mg/ml), and xylazine (2.85 mg/ml). Polyethylenecatheters (PE-50) were placed into the abdominal aorta via the femoralartery to allow rapid withdrawal of arterial blood. A second catheterwas placed in the contralateral femoral artery to monitor bloodpressure, and a third catheter was placed in one of the femoral veinsfor infusion of test materials. Catheters were tunneled subcutaneously,exteriorized through the tail, and flushed with approximately 100 μL ofnormal saline. Animals were allowed to recover from the procedure andremained in their cages for 24 hours before being used in experiments.One femoral artery catheter was connected, through a stopcock, to apressure transducer (UFI model 1050, Morro, Calif.), and arterialpressure was sampled continuously at 100 Hz using a MP100WSW datacollection system (BIOPAC Systems, Inc., Goleta, Calif.). The data werestored in digital form for subsequent off-line analysis.

Mean arterial pressures before and during the exchange transfusion areshown in FIG. 18. All solutions demonstrated significant vasoactivityexcept the PEG-hemoglobin, whose K* value is essentially identical tothat of red blood cells (see FIG. 17).

Based on the data obtained in these experiments, it is contemplated thatautoregulation occurs as a result of oversupply of oxygen due tofacilitated diffusion by cell-free oxygen carriers. The amount of O₂delivered should be the greatest for those solutions that show thegreatest vasoactivity. In vivo experiments of 50% exchange transfusionin a rat are consistent with this theory in that the increase in meanarterial pressure corresponds roughly with the estimated diffusionconstant, K*. Thus K* appears to be the key parameter to use to optimizethe characteristics of a potential red cell substitute.

EXAMPLE 17 Other Hemoglobin Preparations

In this Example, additional hemoglobin preparations are described. Thesepreparations may be modified to provide blood substitutes with thedesirable properties of high oxygen affinity, high oncotic pressure, andrelatively high viscosity (i.e., at least half that of blood).

A. Preparation of Human Hemoglobin A₀

In this experiment, the human hemoglobin A₀ of Christensen et al.(Christensen et al., J. Biochem. Biophys. Meth. 17: 143-154 [1988]) isprepared.

One unit of outdated, packed cells is washed three times in 500 mlplastic centrifuge bottles with sterile 0.9% saline. The wash solutionand the buffy coat are removed with aspiration. The packed cells aremixed with 2.5 volumes of distilled water and centrifuged at 20,000×gfor 1 hour. The supernatant is removed and passed through a mixed-bedion-exchange resin (Bio-Rex RG501-X8, Bio-Rad, Richmond, Calif.) in acolumn. The iso-ionic effluent is passed through 0.22 μm filters(Millipore Millistack 40, Bedford, Mass.) into sterile containers.

For larger quantities of stroma-free hemoglobin: 8 units of packed cellsare washed as above and hemolysis occurs in the cold, overnight. Thelysate is transferred into 600 ml transfer packs (Fenwal 4B2024,Deerfield, Ill.) and spun for 6 hours at 3500×g. Approximately 1/3 ofthe supernatant hemoglobin solution is then removed with a plasmaextractor and passed through the mixed-bed resin until the conductivityis ˜15 μmhos. This process requires 1 kg of resin which is mostconveniently packed into three columns. The solutions are again placedinto transfer packs and centrifuged for 4 hours at 3500×g. Thesupernatants are filtered through a 0.22 μm disposable filter unit(Millipore Millistack, MSG05CHZ), and the filtrate is collected intosterile transfer packs and stored at 4 C for chromatography. Long-termstorage is best achieved by freezing, in bulk, at −80° C.

Stroma-free hemoglobin solutions containing 10-20 g of hemoglobin areequilibrated with 0.05 M Tris-HCl at pH 8.5. This can be done as usualby dialysis or by buffer exchange and gel exclusion columns. However,since these solutions are isoionic, it is more convenient to merelydilute them with an equal volume of 0.1 M Tris-HCl at pH 8.5.Chromatography is performed with a preparative HPLC (Waters Delta-Prep3000). The sample (˜250-500 ml, 4-10 g/dl) is applied to a stainlesssteel column prepacked with QMA-Acell (Waters), previously equilibratedwith 1-2 liters of buffer A (0.05 M Tris-HCl, pH 8.5) at a flow rate of80 ml/min. The chromatogram is developed with a linear gradient of 0.05M Tris-HCl, pH 6.5, as the reserve (buffer B) at the same flow rate. ThepH change is linear from 10% to 90% buffer B, during which allhemoglobin species are eluted. Separations are complete in 50 minutes atwhich time the pH of the effluent buffer is 7.2. Buffer B is the run foran additional 10 minutes to insure complete elution of the samples, andthe column is re-equilibrated with 1 liter buffer A preparatory to asubsequent separation. It is possible to process up to 20 g on onecolumn; however, this appears to be an overload. The column is purgeddaily with 1 liter of 0.1 M Tris-HCl, pH 7.4, in 0.1 M NaCl. Onstanding, it is equilibrated with 70% ethanol.

Peak detection using the preparative cell (2.1 mm pathlength) is at 510nm and/or 600 nm. The latter wavelength is necessary for the higherconcentrations and to amplify the signal due to methemoglobin. The majorfraction of Hb-A₀ is collected to avoid the collection of methemoglobinat the leading edge and the contamination of the minor hemoglobincomponents at the trailing edge. The fraction is collected into a 2liter sterile transfer pack placed in an ice bucket and transferredaseptically into a sterile 2 liter Amicon concentrator (Model 2000B,Danvers, Mass.) equipped with a TMIO membrane (10,000 Da cut-off) filterand the volume reduced to about 10% of the eluate volume at 4° C.

B. Cross-Linking Reactions to Lower P50

In this experiment, various cross-linking methods are tested for theirability to lower P50. In one experiment, the method of Walder et al.(Walder et al. Biochem., 18:4265-4270 [1979]) to producebis(3,5-dibromosalicyl) fumarate (DBBF) and bis(3,5-dibromosalicyl)succinate (DBBS) (β82-β82) is used. Chemical modifications of humanhemoglobin are carried out in 6 g/dl solutions of cell-freeoxyhemoglobin in 0.05 M sodium phosphate, or in 0.05 M Bistris-HCl, pH7.2. Incubations are for 2 hours at 37° C. in a water bath shaker.Reactions are terminated by quenching with glycine.

In another experiment, the method of Manning and Manning (Manning andManning Biochem., 27:6640-6644 [1988]), in which hemoglobin in the Rstate is cross-linked with glycolaldehyde. In this experiment, thehemoglobin concentration varied from 45 to 360 μM in 50 mM potassiumphosphate buffer, pH 7.3. HbCO is used. Glycolaldehyde is added to afinal concentration of 50 mM. The cross-linking is performed at roomtemperature for 4.5 hours, and the hemoglobin derivative is thendialyzed extensively against 50 mM Tris-acetate, pH 7.3.

In yet another experiment, diisothiocyanatobenzenesulfonate (DIBS) isused to cross-link hemoglobin, according to the method of Manning et al.(Manning et al., PNAS 88:3329-3333 [1991]). Hemoglobin solutions (200 μMin the deoxygenated state, usually 3-5 μmoles) are treated with a10-fold molar excess of the crosslinking agent DIBS. The solution isincubated at 25° C. in 0.1 M potassium phosphate, pH 7.2, for 15 min.The reaction is terminated by adding glycylglycine in 30-fold molarexcess; a further incubation for 15 min. is then performed. The solutionis dialyzed at 4° C. against the buffer used for the subsequentchromatographic step. The crosslinked hemoglobin (total 200-250 mg) isapplied to a Whatman DE-52 column (2×30 cm) and eluted with a lineargradient of 50 mM Tris acetate from pH 8.3 to pH 6.3 (500 ml of each).For removal of the most adherent components, the column is furthereluted with 500 ml of the pH 6.3 buffer. Recovery of hemoglobin from thecolumn is 80-95%. For preparative purposes, the cross-linked hemoglobinis passed through a mixed bed resin.

In another experiment, the method of Kluger et al. (Kluger et al.,Biochem., 31:7551-7559 [1992]) is used to cross-link hemoglobin withtrimesoyl tris(methyl phosphate) (β82-β82). In this experiment, chemicalmodifications of hemoglobin are done using hemolysate diluted with 0.1 MBis-Tris-HCl buffer at pH 7.2 to a final concentration of hemoglobintetramer of 1 mM Hb. The final concentration of cross-linking reagent is2 mM in 0.1 M buffer. During the initial phases of this study, thereactions are kept at 35° C. for 2-3 hours with hemoglobin in the COform. To improve yield and to destroy any viral contaminants, thereactions are carried out at 60° C. Reagent is infused at roomtemperature into the 60° C. hemoglobin solution over a period of 30-60min with a total reaction time of up to 3 hours. Reagents and lowmolecular weight byproducts are then removed by gel filtration withSephadex G-25 columns.

In yet another experiment, dicarboxylic acid bis(methyl phosphates)(fumaryl & isophthalyl) (β82-β82) is used according to the method ofJones et al. (Jones et al., Biochem., 32:215-223 [1993]). Chemicalmodifications of hemoglobin are done using hemolysate diluted with 0.1 Mbis-tris-HCl buffer at pH 7.2 to 1 mM Hb (tetramer) and cross-linkingreagent at between 2 mM and 5 mM. The temperature of the reaction iseither 35° C. or 60° C., and the duration of the reaction is 2-3 hours.At the higher temperature, the cross-linking reagent is added slowly byinfusion over ½ to 2 hours. The reactions are run with hemoglobin in thecarbon monoxide form (HbCO). The cross-linking reagents are removed bygel filtration through Sephadex G-25.

C. Hemoglobin Conjugates

In this set of experiments, hemoglobin conjugates are produced usingvarious methods.

1. Hemoglobin Conjugated to Polyoxyethylene

First, 1 ml of a solution containing 1.0 M dibasic phosphate (Na₂HPO₄)and 1.0 M bicarbonate (NaHCO₃) are added to 10 ml of a 9 g/dl hemoglobinsolution at 4° C. with gentle stirring. Then, 1 g ofN-hydroxysuccinimidyl ester of methoxypoly(ethylene glycol) propionicacid, molecular weight 5,000 Da (M-SPA-5000, Shearwater Polymers,Huntsville, Ala.) is added to the solution over a 2 minute period withcontinued stirring. Temperature and pH are monitored throughout thereaction. Addition of the activated polyoxyethylene caused a decrease insolution pH. Approximately 10 mg amounts of solid sodium carbonate(Na₂CO₃) are added to the mixture to maintain the pH in the range8.5-9.5. After 4 hours, the reaction mixture is transferred into 30,000MW dialysis bags (Spectra/Por, Spectrum Medical Industries, Houston,Tex.) and extensively dialyzed against 0.1 M phosphate buffer, pH 7.4.

2. Hemoglobin Conjugated to Polyoxyethylene

In this experiment, the method of Leonard and Dellacherie (Leonard andDellacherie, Biochim. Biophys. Acta 791: 219-225 [1984]) is used.

Activated polyethylene glycol, monomethoxypolyoxyethylenesuccinimidylester (MPSE), MW=5,000 Da, is reacted with stroma-free oxyhemoglobin.1.5 ml of 10 g/dl hemoglobin solution are added to 2 ml 0.1 M phosphatebuffer, water, or 0.1 M NaCl solution. When necessary, pH is adjusted tothe desired value (5.7-7.8) by adding small amounts of 0.1 M NaOH or 0.1M HCl. Then MPSE is added (20-30 mol MPSE per mol hemoglobin tetramer).The reaction mixtures are stirred at 6° C. for 2 hours and then analyzedby gel permeation chromatography on AcA 44 Ultrogel (linearfractionation range 10,000-130,000; exclusion limit 200,000) in 0.05 Mphosphate buffer (pH 7.2) at 6° C. The reactions are considered completewhen the free hemoglobin peak disappeared from the gel permeationchromatograms.

3. Hemoglobin-Polyethylene Glycol Conjugate In this experiment, themethod of Zalipsky et al. (Zalipsky et al., In Polymeric Drugs and DrugDelivery Systems (Dumm, R. L. and Ottenbrite, R. M., eds) pp. 91-100,American Chemical Society, Washington, D.C. 91-100 [1991]) is used.

A. Methoxypoly(ethylene glycol)-N-succinimidyl carbonate (SC-PEG), MW2,000-6,000 Da (1 g, ˜0.2 mmol) is added to a stirred solution of bovineoxyhemoglobin (0.1 g, ˜1.5×10⁻⁶ mol) in 0.1 M sodium phosphate buffer,pH 7.8 (60 ml). Sodium hydroxide (0.5 N) is used to maintain pH 7.8 for30 minutes. The excess free PEG is removed by diafiltration using 50 mMphosphate buffered saline.

B. Methoxypoly(ethylene glycol)-N-succinimidyl carbonate (SC-PEG), MW2,000-6,000 Da (1 g, ˜0.2 mmol) is added to a stirred solution of bovineoxyhemoglobin (0.1 g, ˜1.5×10⁻⁶ mol) in 0.1 M sodium borate buffer, pH9.2. Sodium hydroxide (0.5 N) is used to maintain pH 9.2 for 30 minutes.The excess free PEG is removed by diafiltration using 50 mM phosphatebuffered saline.

4. Hemoglobin-Polyethylene Glycol Conjugate

In this experiment, the methods of Xue and Wong are used (Xue and Wong,Meth. Enz., 231: 308-323 [1994]) to produce hemoglobin-polyethyleneglycol conjugates.

First, activation of PEG: Bis(succinimidyl succinate) is performed. PEG(200 g; 0.059 mol, average 3400 MW; Nippon Oil and Fats Co. Ltd., Tokyo,Japan) is dissolved in 200 ml of dimethylformamide at 100° C., and 15 gof succinic anhydride (0.15 mol) is added. The mixture is stirred for 3hours at 100° C. The dimethylformamide solution is cooled to roomtemperature and poured into 1 liter of ethyl ether. The resulting PEGester of succinic acid is filtered through a glass filter and washedwith ethyl ether. The ester is then dried under vacuum conditions at 40°C. The weight of the product is about 197 g (93% yield).

To activate the succinyl groups on PEG, 197 g of the PEG ester ofsuccinic acid (0.055 mol) is dissolved in 200 ml of dimethylformamide,after which 13 g of N-hydroxysuccinimide (0.11 mol) and 23 g ofdicyclohexylcarbodiimide (0.22 mol) are added. The solution is stirredvigorously overnight at 30° C. The precipitate of dicyclohexylurea isfiltered out and the filtrate is poured into 1 liter of ethyl ether. Thepolyethylene glycol bis(succinimidyl succinate) formed is isolated,washed with ethyl ether repeatedly, and dried under vacuum conditions at40° C. The weight of the product is about 196 g, representing a yieldfrom PEG of 87%.

The purity and the degree of imidylation of polyethylene glycolbis(succinimidyl succinate) may be estimated by nuclear magneticresonance using tetramethylsilane as standard (O ppm) and chloroform-d,as solvent. Similar procedures may be used for the electrophilicactivation of monomethoxypolyethylene glycol.

Next, the activated PEG is conjugated to hemoglobin, with the followingprocedure being carried out at 4° C. First, 0.95 g (0.25 mmol) ofpolyethylene glycol bis(succinimidyl succinate) is added to 100 ml of a0.25 mM Hb solution in 0.1 M sodium phosphate, pH 7.4 and the reactioncontinued for 1 hour. The solution is concentrated by ultrafiltration onan Amicon XM100 membrane. An electrolyte solution is then added and theconcentration process repeated. By repeating this concentrationprocedure three times, unreacted PEG and other low molecular weightcompounds are removed.

Then, the PEG-Hb is stabilized by taking advantage of the ester bondbetween PEG and succinic acid in PEG-Hb, which is labile to hydrolysis.One approach to increase the stability of the bond between PEG and Hb isto remove the labile ester linkage between the polyethylene moiety andthe terminal carboxyls by oxidizing both terminal alcoholic groups ofPEG to carboxylic groups through the use of a metal catalyst, to yield-carboxymethyl-ω-carboxymethoxylpolyoxyethylene, which is activated andcoupled to pyridoxalated Hb as in the case of PEG. The resultantconjugate is designated “stabilized hemoglobin.”

In addition, monomethoxypolyoxyethlylene-hemoglobin is produced. PEG hastwo hydroxyl groups at the two termini. When these are derivatized intofunctional groups capable of reacting with Hb, the presence of tworeactive groups on the same polymer makes possible crosslinkingreactions. Such cross-linking is abolished by blocking one of the twotermini, as in the case of monomethoxypolyoxyethylene (MPOE).

To produce MPOE, 80 g (4 mmol) of MW 5000 MPOE from Aldrich (Milwaukee,Wis.) is dissolved in tetrahydrofuran (300 ml) and treated withnaphthalene sodium under nitrogen at room temperature for 3 hours. ThenBrCH₂COOC₂H₅ (1.4 ml; 12 mmol) is added dropwise with stirring. After 4hours of reaction, the ethyl ester obtained is precipitated with ether,dried, dissolved in water, and saponified with 0.1 N NaOH at 55° C. for24 hours to yield MPOE-carboxylic acid (MPOE-O—CH₂COOH). The solution isthen acidified with 1 N HCl down to pH 2.5, and the polymer taken upwith chloroform. After several washings with water, the organic layer isdried over MgSO₄ and treated with charcoal. The MPOE-carboxylic acid isprecipitated with dry ether, filtered, and dried under vacuum. This runof operations is repeated until the potentiometric titration gives aconstant value for the quantity of fixed COOH.

The MPOE-carboxylic acid (5 g; 1 mmol) is dissolved in dry ethyl acetate(60 ml) and activated by N-hydroxysuccinimide (0.15 g; 1.25 mmol) anddicyclohexylcarbodiimide (0.26 g; 1.25 mmol) at 30° C. for 15 hours.Dicyclohexylurea is removed by filtration and the polymer precipitatedwith dry ether is taken up with chloroform and crystallized from thissolution by dropwise addition of ether at 0° C. This procedure isrepeated several times until the spectrophotometric analysis ofsuccinimidyl groups gave a constant value.

Coupling to hemoglobin is performed at 5° C. by diluting 1.5 ml of a 10g/dl hemoglobin solution with 2 ml 0.1 M phosphate buffer, pH 5.8, and300 mg of MPOE-carboxylic succinimidyl ester is added under stirring.The reaction mixture is stirred at 6° C. for 2 hours and analyzed by gelpermeation chromatography on Ultrogel AcA 34 (linear fractionation rangeMW 20,000-350,000; exclusion limit 750 000) in 0.05 M phosphate buffer(pH 7.2) at 6° C.

5. Hemoglobin-Dextran Conjugate

In this experiment, hemoglobin-dextran conjugates are produced accordingto the various methods of Kue and Wong (Xue and Wong, Meth. Enz., 231:308-323 [1994]).

Synthesis by Alkylation

In this method, the dextran (Dx) is first derivatized with cyanogenbromide and diaminoethane to contain a free amino group, which isacylated with bromoacetyl bromide. The bromoacetyl function in turnalkylates the sulfhydryl of the β93 cysteine on Hb:

-   -   Dx+CNBR+diaminoethane→aminoethyl-Dx    -   Aminoethyl-Dx+bromoacetyl bromide→Dx-NHCOCH₂Br    -   Dx-NHCOCH₂Br+HS-Hb→Dx-NHCOCH₂—S-Hb

In a typical preparation, 1.5 g of cyanogen bromide is dissolved in 15ml of acetonitrile and added to 10 g of dextran (MW 20,000) in 375 ml ofwater. The pH is maintained at 10.8 for 5 min by the addition of 1 MNaOH; the pH is then lowered to about 2.0-2.5 with concentrated HCl.After stirring for 1 min, 15 ml of diaminoethane is added along withsufficient HCl to prevent the pH from exceeding 9.5. The final pH isadjusted to 9.5. After standing overnight at 4° C., the mixture isthoroughly dialyzed against distilled water using a Millipore(Marlborough, Mass.) Pellicon dialyzer and lyophilized. Theaminoethyl-Dx so obtained is dissolved in 250 ml of 0.1 M sodiumphosphate, pH 7.0, and 15 ml of bromoacetyl bromide is added through aPasteur pipette with a finely drawn capillary tip, accompanied byvigorous stirring over a period of 2 hours. Throughout, the pH ismaintained at 7.0 with the use of a pH-stat and addition of 1 M NaOH.Afterward, the mixture is dialyzed thoroughly against distilled waterand is lyophilized to yield about 7 g of the Dx-NHCOCH₂Br (Br-dextran).The bromine content of the Br-dextran is in the range of 9-11 glucoseresidues per bromine atom.

To couple hemoglobin to dextran, 3.3 g of Br-dextran is dissolved in 100ml of 6 g/dl hemoglobin solution in 0.1 M sodium bicarbonate, pH 9.5.The coupling reaction is allowed to proceed with constant mixing at 4°C. To determine the yield of Dx-NHCOCH₂—S-Hb (Dx-Hb), 0.1 ml of thereaction mixture is applied to a Sephadex G-75 column equilibrated with0.05 M phosphate buffer, pH 7.5, and eluted with the same buffer, at aflow rate of 40 ml/hr. The hemoglobin content of the eluant fractions isdetermined by absorbance at 415 nm, and the proportions of the fastermigrating Dx-Hb peak and the slower migrating Hb peak were given by theareas under these peaks. After 2 days the formation of the Dx-Hbconjugate is essentially complete.

Synthesis by Dialdehyde

Ten ml of a 12% aqueous solution of sodium periodate is added to 100 mlof a 10% aqueous solution of dextran, and the mixture is left overnightin the dark at 4° C. A 3% solution of sodium bisulfite is added untilthe mixture turned brown and then, once again, colorless. The mixture isdialyzed against distilled water to yield the dextran dialdehydesolution. It is then added to 2 volumes of 3 g/dl stroma-free hemoglobinin 0.3 M sodium bicarbonate buffer, pH 9.5; coupling of hemoglobin todextran is allowed to proceed overnight at 4° C. The Dx-Hb complexformed is separated from uncoupled hemoglobin by means of chromatographyon a Sephadex G-75 column.

Coupling of Hb to Dx-dialdehyde is pH dependent. When coupling isperformed by dissolving 100 mg Dx-dialdehyde in 1 ml of 0.6 M sodiumborate buffer and mixing with 1.8 ml of 10 g/dl Hb at 6° C., many labileimine linkages are formed at pH<9.6, and the conjugates have a highmolecular weight, ranging to above 100,000. At higher pH, the majorproduct has a lower molecular weight range (70,000>MW>100,000) andlikely consists of a 1:1 complex between Dx and Hb, which only slowlyconverts to higher molecular weight forms. When this conjugate is formedat pH 9.8 and reduced at pH 7.2 for 30 min with excess NaBH₄ (2 mol permole of initial aldehyde) dissolved in 1 mM NaOH, only the α chain ofhemoglobin is found to be modified by Dx. Coupling of Hb toDx-dialdehyde also proceeds much more rapidly at higher pH, requiringless than 1 hour for completion at pH 10 and only 1.5 hours at pH 9.7,but 6 hours at pH 9.5 and 23 hours at pH 9.1. When prepared at pH 9.75,the oxygen P50 for Dx-Hb is 10.1 mm Hg when the conjugate is allowed toform for 1 hour prior to NaBH₄ reduction, 9.5 mm Hg when allowed to formfor 4 hours, and 8.1 when allowed to form for 18 hours.

6. Hemoglobin Conjugation to SF-DX and P-Dx

In this experiment, hemoglobin is conjugated with SF-DX and P-Dx.Dextran-sulfate (SF-Dx) and dextran-phosphate (P-Dx) (MW 40,000) aretreated with sodium periodate to generate the dialdehydyl derivatives,which are in turn coupled to the amino groups on hemoglobin and arefurther stabilized by reduction with sodium borohydride, as describedabove in the synthesis of Dx-Hb from Dx-dialdehyde.

7. Hemoglobin Conjugation to Dextran-Benzene Hexacarboxylate (Dx-BHC)

In this experiment, hemoglobin is conjugated with dextran-benzenehexacarboxylate (Dx-BHC). Aminopropyl-Dx is prepared 35 by dissolving 5g of dextran in 7.5 ml of 25% aqueous Zn(BF₄)₂ and 5 ml of water.Epichlorohydrin (25 ml) is added with vigorous stirring; the mixture isallowed to react for 3 hours at 80° C. and subsequently overnight atroom temperature. The polymer is precipitated by pouring the solutiondropwise into acetone, filtered, and dried under reduced pressure. Theresulting dextran derivative has the structure of Dx-O—CH₂CH(OH)CH₂Cl.This product (4.1 g containing 3% Cl) is purified by repeateddissolution in water and precipitation by acetone and methanol. Thechlorine atom is subsequently replaced by an amino group by dissolvingthe compound in 60 ml of H₂O and 20 ml of 14 M aqueous ammonia. Thesolution is stirred for 20 hours at room temperature and then poureddropwise into 1 liter of methanol. The resulting precipitate ofaminopropyl-Dx (3-amino-2-hydroxypropyl ether of dextran) is filtered,washed with acetone, and dried under reduced pressure. The yield at thisstage is about 3.5 g.

Benzene hexacarboxylic acid is coupled to aminopropyl-Dx to form Dx-BHCthrough the use of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (EDCI) as condensing agent. Because benzene hexacarboxylicacid has six carboxylic acid groups, reaction with an amino group onaminopropyl-Dx still leaves it with up to five carboxylic acid groups.One of these may be linked to an amino group on Hb through further useof the water-soluble EDCI as condensing agent.

8. Hydroxyethyl Starch-Hemoglobin-Conjugate

In this experiment, the method of Xue and Wong, (Xue and Wong, Meth.Enz., 231: 308-323 [1994]) is used to produce hydroxyethylstarch-hemoglobin conjugates.

To prepare for conjugation to hemoglobin, the hydroxyethyl starch (Hs)is first converted to aminoethyl-Hs. In a typical preparation, 1.5 g ofcyanogen bromide is dissolved in 15 ml of acetonitrile and added to 500ml of 2% Hs solution. The pH of the solution is maintained at 10.8 for5-10 min by the addition of 1 M NaOH solution. The pH is then lowered to2.0-2.5 with concentrated HCl, and 10 ml of diaminoethane is added alongwith additional HCl to prevent the pH from exceeding 9.5. The final pHis adjusted to 9.5 and the solution is allowed to stand overnight at 4°C. before being dialyzed against deionized water. The ratio of cyanogenbromide/diaminoethane to Hs can be varied, allowing the synthesis ofaminoethyl-Hs in which from 7 to 20% of the glucose residues in thestarting polymer are substituted.

Aldehyde-substituted Hs is prepared by reaction of the aminoethyl-Hswith glutaraldehyde. In a typical reaction, 500 ml of dialyzed solutionof aminoethyl-Hs is treated with 2 g of sodium bicarbonate to give asolution 2% in Hs and approximately 0.05 M in bicarbonate. Then 5 ml of50% glutaraldehyde solution is added to the solution, which is stirredat room temperature for 2 hours before dialysis.

Hemoglobin is employed as a freeze-dried solid under carbon monoxide.This is reconstituted under argon using deoxygenated deionized water at4° C. to give a solution with approximately 2.5 g Hb per ml. In atypical reaction, 500 ml of dialyzed solution of thealdehyde-substituted Hs is treated with sodium bicarbonate to give 500ml of solution approximately 2% in Hs and 0.1 M in bicarbonate,hemoglobin solution (25 ml) is added and the reaction is stirred at roomtemperature for 4 hours, after which time get filtration on SephadexG-150 indicates that no unmodified hemoglobin remains. Sodiumborohydride (1.0 g) is then added to the solution, which is stirred fora further 2 hours at room temperature. The Hs-Hb is dialyzed using anAmicon (Danvers, Mass.) ultrafiltration unit with a 100,000 molecularweight cutoff cartridge to enable the removal of any trace of unmodifiedhemoglobin. Glucose (10 g) is added to the solution prior tofreeze-drying and storage under carbon monoxide at 4° C.

9. An Alternative Method for Producing Hydroxyethyl Starch-HemoglobinConjugates

In this experiment, another method described by Xue and Wong (Xue andWong, Meth. Enz., 231: 308-323 [1994]) was used to produce hydroxyethylstarch-hemoglobin conjugates.

Hydroxyethyl starch-hemoglobin-conjugate (Hs-Hb) can be synthesized fromHs-dialdehyde as follows. 0.03 equivalents of Hs are dissolved in 250 mlof water and treated with 0.028 mol of sodium periodate for 12 hours at5° C. in the dark. The solution is dialyzed until ion free. The percentoxidation may be determined using a calorimetric method. The solution isbuffered to pH 8.0 by addition of sodium bicarbonate, cooled to 5° C.,and treated with 5 g of carbonmonoxyhemoglobin. The reaction is allowedto proceed for 18 hours at room temperature or until gel filtrationindicates complete modification of hemoglobin. The solution is dialyzedagainst 1% ammonium carbonate and freeze-dried in the presence ofglucose.

10. Hemoglobin-Inulin Conjugate

In this experiment, a method described by Xue and Wong (Xue and Wong,Meth. Enz., 231: 308-323 [1994]) is used to produce hemoglobin-inulinconjugates.

To synthesize the inulin-hemoglobin (In-Hb) conjugate, inulin is firstsuccinylated by reacting with succinic anhydride inN,N-dimethylformamide at 100° C. for 2 hours. Subsequently, thesuccinylated inulin is linked to N-hydroxysuccinimide at roomtemperature overnight using dicyclohexylcarbodiimide as condensationagent in N,N-dimethylformamide. Hemoglobin is allowed to react with a10-fold molar excess of the N-hydroxysuccinimide-activated inulin in 0.1M Tris buffer, pH 7.0, at 4° C. for 1 hour to yield In-Hb, which ispurified with an Amicon PM30 membrane filter until the unreacted inulinand other low molecular weight compounds are removed.

By controlling the succinic anhydride/inulin ratio, the number ofN-hydroxysuccinimide-activated succinyl groups on the inulin can bevaried. A low density of such groups gives rise to a 82,000 MW In-Hbconjugate, whereas higher densities produce cross-linked In-Hb rangingup to above 300,000 MW.

11. An Alternative Method to Produce Hemoglobin-Inulin Conjugates

In this experiment, the method of Iwasaki et al. (Iwasaki et al.,Biochem. Biophys. Res. Comm., 113: 513-518 [1983]) is used to producehemoglobin-inulin conjugates.

The N-hydroxysuccinimidyl ester of inulin was reacted with oxyhemoglobinin 0.1 M tris buffer (pH 7.0) at 4° C. for one hour. The reactionmixture was analyzed with a JASCO Trirotor HPLC apparatus equipped witha TSK G3000 SW column. The modified hemoglobin solution was purifiedwith an Amicon PM 30 membrane filter until the unreacted inulin andother low molecular weight compounds are no longer detected.

12. Hemoglobin-Polyvinylpyrrolidone Conjugate

In this experiment, the method of Xue and Wong (Xue and Wong, Meth. Enz,231: 308-323 [1994]) was used to produce hemoglobin-polyvinylpyrrolidoneconjugates.

Synthesis of Activated PVP

First, 50 g of polyvinylpyrrolidone (PVP) (MW 25,000-35,000) isdissolved in 1 liter of 0.25 N NaOH and heated at 140° C. for 42 hoursunder nitrogen in an autoclave to bring about partial hydrolysis. It isthen adjusted to pH 5 with concentrated HCl and ultrafiltered through anAmicon UM10 membrane to remove salts. Water is removed throughazeotropic distillation with benzene, and the extent of hydrolysis isdetermined by titration of the secondary amino groups. To blockade theseamino groups, 50 g of the partially hydrolyzed PVP is dissolved in 300ml of dichloromethane/dimethylformamide (1:1) and mixed with 0.5 M ofacetic acid anhydride. It is left at room temperature for 1 hour andrefluxed for 4 hours. Evacuated to about 100 ml, the solution is addeddropwise into ethyl ether under strong stirring. The acetylated PVPprecipitate is filtered, washed with ether, and dried to constant weightunder vacuum over phosphorus pentoxide.

To activate its carboxyl groups, 50 g of acetylated PVP dissolved in 500ml of dichloromethane/dimethylformamide (1:1) is mixed at 0° C. with15.47 g of N-hydroxysuccinimide followed with a solution of 27.75 gdicyclohexylcarbodiimide in 50 ml of dichloromethane. The solution isstirred at 0° C. for 14 hours before centrifugation to remove thedicyclohexylurea. The supernatant solution (about 300 ml) is addeddropwise into 5 liters of cold ether under strong stirring. The whiteprecipitate is filtered, washed repeatedly with ether, and dried in thecold over phosphorus pentoxide.

Binding of Hemoglobin to Activated PVP

Hemoglobin (27 g) is dissolved in 1 liter of 5% sodium carbonate andtreated at 4° C. with 40 g of activated PVP for 24 hours with stirring.The preparation is lyophilized and redissolved in 300 ml of distilledwater. After a 20-fold volume diafiltration, it is again lyophilized.

From the above, it should be evident that the present invention providesoptimal blood substitute compositions comprising mixtures ofoxygen-carrying and non-oxygen carrying plasma expanders and methods forthe use thereof. These compositions and methods allow for the productionof relatively inexpensive products that are more effective thancurrently available compositions.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inhematology, surgical science, transfusion medicine, transplantation, orany related fields are intended to be within the scope of the followingclaims.

1-44. (canceled)
 45. An aqueous cell-free composition for administrationto a subject, comprising polyalkylene oxide modified hemoglobin in anaqueous solution, wherein said modified hemoglobin has a molecularradius larger than native hemoglobin, wherein said composition has aviscosity at least half that of blood, and an oncotic pressure higherthan that of plasma, and wherein said polyalkylene oxide is covalentlyattached to cysteine residues on the hemoglobin.
 46. The compositionaccording to claim 45, wherein P50 of said composition is equal to orlower than that of blood.
 47. The composition according to claim 45,wherein the modified hemoglobin is human hemoglobin.
 48. The compositionaccording to claim 45, wherein the viscosity is from 2 to 4.5 cPs. 49.The composition according to claim 48, wherein the viscosity is at least2 cPs.
 50. The composition according to claim 49, wherein the viscosityis 2.5 cPs.
 51. The composition according to claim 45, wherein themodified hemoglobin concentration is greater than 4.0 g/dl.
 52. Thecomposition according to claim 45, wherein P50 of said composition isless than 28 mm Hg.
 53. The composition according to claim 45, whereinthe polyalkylene oxide is polyethylene glycol.
 54. The compositionaccording to claim 45, wherein the oncotic pressure is greater than 25mm Hg.
 55. The composition according to claim 45, wherein the oncoticpressure is from 20 to 60 mm Hg.
 56. The composition according to claim45, wherein said polyalkylene oxide is covalently attached to sulfhydrylgroups of said cysteine residues.
 57. An aqueous cell-free compositionfor administration to a subject, comprising polyethylene glycolconjugated to sulfhydryl groups on human hemoglobin to form a modifiedhemoglobin in an aqueous solution, wherein said modified hemoglobin hasa molecular radius larger than native hemoglobin, wherein saidcomposition has a viscosity from 2 to 4.5 cPs, an oncotic pressure from20 to 60 mm Hg, and a modified hemoglobin concentration greater than 4.0g/dl.
 58. The composition according to claim 57, wherein said sulfhydrylgroups are on cysteine residues.